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Sample records for cell receptor binding

  1. Binding of /sup 125/I-labeled reovirus to cell surface receptors

    Energy Technology Data Exchange (ETDEWEB)

    Epstein, R.L.; Powers, M.L.; Rogart, R.B.; Weiner, H.L.

    1984-02-01

    Quantitative studies of /sup 125/I-labeled reovirus binding at equilibrium to several cell types was studied, including (1) murine L cell fibroblasts; (2) murine splenic T lymphocytes; (3) YAC cells, a murine lymphoma cell line; and (4) R1.1 cells, a murine thymoma cell line. Competition and saturation studies demonstrated (1) specific, saturable, high-affinity binding of reovirus types 1 and 3 to nonidentical receptors on L cell fibroblasts; (2) high-affinity binding of type 3 reovirus to murine splenic lymphocytes and R1.1 cells; (3) low-affinity binding of reovirus type 1 to lymphocytes and R1.1 cells; and (4) no significant binding of either serotype to YAC cells. Differences in the binding characteristics of the two reovirus serotypes to L cell fibroblasts were found to be a property of the viral hemagglutinin, as demonstrated using a recombinant viral clone. The equilibrium dissociation constant (Kd) for viral binding was of extremely high affinity (Kd in the range of 0.5 nM), and was slowly reversible. Experiments demonstrated temperature and pH dependence of reovirus binding and receptor modification studies using pronase, neuraminidase, and various sugars confirmed previous studies that reovirus receptors are predominantly protein in structure. The reovirus receptor site density was in the range of 2-8 X 10(4) sites/cell. These studies demonstrate that the pseudo-first-order kinetic model for ligand-receptor interactions provides a useful model for studying interactions of viral particles with membrane viral receptors. They also suggest that one cell may have distinct receptor sites for two serotypes of the same virus, and that one viral serotype may bind with different kinetics depending on the cell type.

  2. RAINBOW TROUT ANDROGEN RECEPTOR ALPHA AND THE HUMAN ANDROGEN RECEPTOR: COMPARISONS IN THE COS WHOLE CELL BINDING ASSAY

    Science.gov (United States)

    Rainbow Trout Androgen Receptor Alpha And Human Androgen Receptor: Comparisons in the COS Whole Cell Binding Assay Mary C. Cardon, L. Earl Gray, Jr. and Vickie S. WilsonU.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle...

  3. Cargo binding promotes KDEL receptor clustering at the mammalian cell surface.

    Science.gov (United States)

    Becker, Björn; Shaebani, M Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J

    2016-06-29

    Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTA(H/KDEL)), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    of receptor function and in turn for the design and development of novel therapeutic compound. Here we show how ligand-receptor interaction can be investigated in situ with high sensitivity on sensor surfaces by total internal reflection fluorescence (TIRF) measurements. A generally applicable method...... for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF...... measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification....

  5. Loss of Glycosaminoglycan Receptor Binding after Mosquito Cell Passage Reduces Chikungunya Virus Infectivity.

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    Dhiraj Acharya

    Full Text Available Chikungunya virus (CHIKV is a mosquito-transmitted alphavirus that can cause fever and chronic arthritis in humans. CHIKV that is generated in mosquito or mammalian cells differs in glycosylation patterns of viral proteins, which may affect its replication and virulence. Herein, we compare replication, pathogenicity, and receptor binding of CHIKV generated in Vero cells (mammal or C6/36 cells (mosquito through a single passage. We demonstrate that mosquito cell-derived CHIKV (CHIKV mos has slower replication than mammalian cell-derived CHIKV (CHIKV vero, when tested in both human and murine cell lines. Consistent with this, CHIKV mos infection in both cell lines produce less cytopathic effects and reduced antiviral responses. In addition, infection in mice show that CHIKV mos produces a lower level of viremia and less severe footpad swelling when compared with CHIKV vero. Interestingly, CHIKV mos has impaired ability to bind to glycosaminoglycan (GAG receptors on mammalian cells. However, sequencing analysis shows that this impairment is not due to a mutation in the CHIKV E2 gene, which encodes for the viral receptor binding protein. Moreover, CHIKV mos progenies can regain GAG receptor binding capability and can replicate similarly to CHIKV vero after a single passage in mammalian cells. Furthermore, CHIKV vero and CHIKV mos no longer differ in replication when N-glycosylation of viral proteins was inhibited by growing these viruses in the presence of tunicamycin. Collectively, these results suggest that N-glycosylation of viral proteins within mosquito cells can result in loss of GAG receptor binding capability of CHIKV and reduction of its infectivity in mammalian cells.

  6. Nuclear thyroid hormone receptor binding in human mononuclear blood cells after goitre resection

    DEFF Research Database (Denmark)

    Kvetny, J; Matzen, L E; Blichert-Toft, M

    1989-01-01

    Nuclear thyroxine and triiodothyronine receptor-binding in human mononuclear blood cells were examined in 14 euthyroid persons prior to and 1, 6, 24 and 53 weeks after goitre resection. One week after resection decreased serum T3 from 1.47 nmol/l to 1.14 nmol/l (P less than 0.05), FT4I from 103 a...

  7. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Behrendt, N; Ploug, M; Patthy, L;

    1991-01-01

    part of the intact receptor, probably including the whole sequence 1-87, and contained N-linked carbohydrate. After detergent phase separation in the Triton X-114 system, the fragment was present in the water phase where its binding activity could be demonstrated in the absence of the rest...... applications in interfering with cell-surface plasmin-mediated proteolysis....

  8. The neural cell adhesion molecule binds to fibroblast growth factor receptor 2

    DEFF Research Database (Denmark)

    Christensen, Claus; Lauridsen, Jes B; Berezin, Vladimir;

    2006-01-01

    The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface...

  9. T cell receptor signaling can directly enhance the avidity of CD28 ligand binding.

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    Mariano Sanchez-Lockhart

    Full Text Available T cell activation takes place in the context of a spatial and kinetic reorganization of cell surface proteins and signaling molecules at the contact site with an antigen presenting cell, termed the immunological synapse. Coordination of the activation, recruitment, and signaling from T cell receptor (TCR in conjunction with adhesion and costimulatory receptors regulates both the initiation and duration of signaling that is required for T cell activation. The costimulatory receptor, CD28, is an essential signaling molecule that determines the quality and quantity of T cell immune responses. Although the functional consequences of CD28 engagement are well described, the molecular mechanisms that regulate CD28 function are largely unknown. Using a micropipet adhesion frequency assay, we show that TCR signaling enhances the direct binding between CD28 and its ligand, CD80. Although CD28 is expressed as a homodimer, soluble recombinant CD28 can only bind ligand monovalently. Our data suggest that the increase in CD28-CD28 binding is mediated through a change in CD28 valency. Molecular dynamic simulations and in vitro mutagenesis indicate that mutations at the base of the CD28 homodimer interface, distal to the ligand-binding site, can induce a change in the orientation of the dimer that allows for bivalent ligand binding. When expressed in T cells, this mutation allows for high avidity CD28-CD80 interactions without TCR signaling. Molecular dynamic simulations also suggest that wild type CD28 can stably adopt a bivalent conformation. These results support a model whereby inside-out signaling from the TCR can enhance CD28 ligand interactions by inducing a change in the CD28 dimer interface to allow for bivalent ligand binding and ultimately the transduction of CD28 costimulatory signals that are required for T cell activation.

  10. Roles of cell and microvillus deformation and receptor-ligand binding kinetics in cell rolling.

    Science.gov (United States)

    Pawar, Parag; Jadhav, Sameer; Eggleton, Charles D; Konstantopoulos, Konstantinos

    2008-10-01

    Polymorphonuclear leukocyte (PMN) recruitment to sites of inflammation is initiated by selectin-mediated PMN tethering and rolling on activated endothelium under flow. Cell rolling is modulated by bulk cell deformation (mesoscale), microvillus deformability (microscale), and receptor-ligand binding kinetics (nanoscale). Selectin-ligand bonds exhibit a catch-slip bond behavior, and their dissociation is governed not only by the force but also by the force history. Whereas previous theoretical models have studied the significance of these three "length scales" in isolation, how their interplay affects cell rolling has yet to be resolved. We therefore developed a three-dimensional computational model that integrates the aforementioned length scales to delineate their relative contributions to PMN rolling. Our simulations predict that the catch-slip bond behavior and to a lesser extent bulk cell deformation are responsible for the shear threshold phenomenon. Cells bearing deformable rather than rigid microvilli roll slower only at high P-selectin site densities and elevated levels of shear (>or=400 s(-1)). The more compliant cells (membrane stiffness=1.2 dyn/cm) rolled slower than cells with a membrane stiffness of 3.0 dyn/cm at shear rates >50 s(-1). In summary, our model demonstrates that cell rolling over a ligand-coated surface is a highly coordinated process characterized by a complex interplay between forces acting on three distinct length scales.

  11. Ligand-receptor binding kinetics in surface plasmon resonance cells: A Monte Carlo analysis

    CERN Document Server

    Carroll, Jacob; Forsten-Williams, Kimberly; Täuber, Uwe C

    2016-01-01

    Surface plasmon resonance (SPR) chips are widely used to measure association and dissociation rates for the binding kinetics between two species of chemicals, e.g., cell receptors and ligands. It is commonly assumed that ligands are spatially well mixed in the SPR region, and hence a mean-field rate equation description is appropriate. This approximation however ignores the spatial fluctuations as well as temporal correlations induced by multiple local rebinding events, which become prominent for slow diffusion rates and high binding affinities. We report detailed Monte Carlo simulations of ligand binding kinetics in an SPR cell subject to laminar flow. We extract the binding and dissociation rates by means of the techniques frequently employed in experimental analysis that are motivated by the mean-field approximation. We find major discrepancies in a wide parameter regime between the thus extracted rates and the known input simulation values. These results underscore the crucial quantitative importance of s...

  12. Receptors from glucocorticoid-sensitive lymphoma cells and two clases of insensitive clones: physical and DNA-binding properties.

    Science.gov (United States)

    Yamamoto, K R; Stampfer, M R; Tomkins, G M

    1974-10-01

    Mouse lymphoma tissue culture cells (S49.1A) are normally killed by dexamethasone, a synthetic glucocorticoid hormone. Dexamethasone-resistant clones have been selected from this line, some of which retain the ability to specifically bind dexamethasone. Addition of [(3)H]dexamethasone to cultures, followed by cell fractionation, reveals that the nuclear transfer of hormone-receptor complexes in some of these variant clones is deficient (nt(-)), while others show increased nuclear transfer (nt(i)) relative to the parental line. Two independently selected members of each class have been studied here, in an effort to elucidate the molecular determinants involved in the receptor-nucleus interaction in vivo. The labeled receptors in cell-free extracts bind to DNA-cellulose, but only after previous incubation of the extract at 20 degrees , similar to the treatment required for cell-free interaction of receptors with nuclei. More importantly, the apparent DNA-binding affinity of the nt(-) receptors is lower than the wild type, whereas the nt(i) receptors bind DNA with an affinity higher than the parental molecules. The parallelism of nuclear and DNA binding, together with the observations that the receptors from the variants have sedimentation properties different from the wild-type cells, lead us to conclude that (i) these variants may contain altered receptor molecules and (ii) DNA is probably the primary nuclear binding site for steroid receptors in vivo.

  13. Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses.

    Science.gov (United States)

    Kaiser, Liselotte; Graveland-Bikker, Johanna; Steuerwald, Dirk; Vanberghem, Mélanie; Herlihy, Kara; Zhang, Shuguang

    2008-10-14

    High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a alpha-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins.

  14. Characterization of human platelet binding of recombinant T cell receptor ligand

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    Meza-Romero Roberto

    2010-11-01

    Full Text Available Abstract Background Recombinant T cell receptor ligands (RTLs are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS. RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE. The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood. Methods We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function. Results Our data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen. Conclusions Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation.

  15. LIGAND-BINDING PROFILE OF THE RAT METABOTROPIC GLUTAMATE-RECEPTOR MGLUR3 EXPRESSED IN A TRANSFECTED CELL-LINE

    NARCIS (Netherlands)

    LAURIE, DJ; DANZEISEN, M; BODDEKE, HWGM; SOMMER, B

    1995-01-01

    A cDNA clone encoding the rat metabotropic glutamate receptor mGluR3 was stably transfected into human embryonic kidney 293 cells. Receptor-expressing cell lines were characterized by centrifugation binding assays using [H-3]glutamate as radioligand. The rank order of affinity was L-glutamate>(1S,3R

  16. A genome-wide signature of glucocorticoid receptor binding in neuronal PC12 cells

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    Polman J Annelies E

    2012-10-01

    Full Text Available Abstract Background Glucocorticoids, secreted by the adrenals in response to stress, profoundly affect structure and plasticity of neurons. Glucocorticoid action in neurons is mediated by glucocorticoid receptors (GR that operate as transcription factors in the regulation of gene expression and either bind directly to genomic glucocorticoid response elements (GREs or indirectly to the genome via interactions with bound transcription factors. These two modes of action, respectively called transactivation and transrepression, result in the regulation of a wide variety of genes important for neuronal function. The objective of the present study was to identify genome-wide glucocorticoid receptor binding sites in neuronal PC12 cells using Chromatin ImmunoPrecipitation combined with next generation sequencing (ChIP-Seq. Results In total we identified 1183 genomic binding sites of GR, the majority of which were novel and not identified in other ChIP-Seq studies on GR binding. More than half (58% of the binding sites contained a GRE. The remaining 42% of the GBS did not harbour a GRE and therefore likely bind GR via an intermediate transcription factor tethering GR to the DNA. While the GRE-containing binding sites were more often located nearby genes involved in general cell functions and processes such as apoptosis, cell motion, protein dimerization activity and vasculature development, the binding sites without a GRE were located nearby genes with a clear role in neuronal processes such as neuron projection morphogenesis, neuron projection regeneration, synaptic transmission and catecholamine biosynthetic process. A closer look at the sequence of the GR binding sites revealed the presence of several motifs for transcription factors that are highly divergent from those previously linked to GR-signaling, including Gabpa, Prrx2, Zfp281, Gata1 and Zbtb3. These transcription factors may represent novel crosstalk partners of GR in a neuronal context

  17. Autoantigenic targets of B-cell receptors derived from chronic lymphocytic leukemias bind to and induce proliferation of leukemic cells.

    Science.gov (United States)

    Zwick, Carsten; Fadle, Natalie; Regitz, Evi; Kemele, Maria; Stilgenbauer, Stephan; Bühler, Andreas; Pfreundschuh, Michael; Preuss, Klaus-Dieter

    2013-06-06

    Antigenic targets of the B-cell receptor (BCR) derived from malignant cells in chronic lymphocytic leukemia (CLL) might play a role in the pathogenesis of this neoplasm. We screened human tissue-derived protein macroarrays with antigen-binding fragments derived from 47 consecutive cases of CLL. An autoantigenic target was identified for 12/47 (25.5%) of the cases, with 3 autoantigens being the target of the BCRs from 2 patients each. Recombinantly expressed autoantigens bound specifically to the CLL cells from which the BCR used for the identification of the respective autoantigen was derived. Moreover, binding of the autoantigen to the respective leukemic cells induced a specific activation and proliferation of these cells. In conclusion, autoantigens are frequent targets of CLL-BCRs. Their specific binding to and induction of proliferation in the respective leukemic cells provide the most convincing evidence to date for the long-time hypothesized role of autoantigens in the pathogenesis of CLL.

  18. Machupo virus glycoprotein determinants for human transferrin receptor 1 binding and cell entry.

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    Sheli R Radoshitzky

    Full Text Available Machupo virus (MACV is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1. TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors.

  19. Deoxyribonucleic acid-binding ability of androgen receptors in whole cells: implications for the actions of androgens and antiandrogens

    NARCIS (Netherlands)

    C.W. Kuil (Cor); E. Mulder (Eppo)

    1996-01-01

    textabstractIn whole cells, the effects of several androgens and antiandrogens on the in the induction of DNA binding for the human wild-type androgen receptor (AR) and a mutant receptor ARL (LNCaP mutation; codon 868, Thr to Ala) were examined and related to the transc

  20. Brownian nanoimaging of interface dynamics and ligand-receptor binding at cell surfaces in 3-D.

    Science.gov (United States)

    Kuznetsov, Igor R; Evans, Evan A

    2013-04-01

    We describe a method for nanoimaging interfacial dynamics and ligand-receptor binding at surfaces of live cells in 3-D. The imaging probe is a 1-μm diameter glass bead confined by a soft laser trap to create a "cloud" of fluctuating states. Using a facile on-line method of video image analysis, the probe displacements are reported at ~10 ms intervals with bare precisions (±SD) of 4-6 nm along the optical axis (elevation) and 2 nm in the transverse directions. We demonstrate how the Brownian distributions are analyzed to characterize the free energy potential of each small probe in 3-D taking into account the blur effect of its motions during CCD image capture. Then, using the approach to image interactions of a labeled probe with lamellae of leukocytic cells spreading on cover-glass substrates, we show that deformations of the soft distribution in probe elevations provide both a sensitive long-range sensor for defining the steric topography of a cell lamella and a fast telemetry for reporting rare events of probe binding with its surface receptors. Invoking established principles of Brownian physics and statistical thermodynamics, we describe an off-line method of super resolution that improves precision of probe separations from a non-reactive steric boundary to ~1 nm.

  1. The retinol esterifying enzyme LRAT supports cell signaling by retinol-binding protein and its receptor STRA6

    OpenAIRE

    Marwarha, Gurdeep; Berry, Daniel C.; Croniger, Colleen M.; Noy, Noa

    2014-01-01

    Vitamin A, retinol, circulates in blood bound to retinol-binding protein (RBP). At some tissues, holo-RBP is recognized by a plasma membrane receptor termed STRA6, which serves a dual role: it mediates transport of retinol from RBP into cells, and it functions as a cytokine receptor that, on binding holo-RBP, activates JAK2/STAT5 signaling. As STAT target genes include SOCS3, an inhibitor of insulin receptor, holo-RBP suppresses insulin responses in STRA6-expressing cells. We have shown previ...

  2. Cytisine binds with similar affinity to nicotinic alpha4beta2 receptors on the cell surface and in homogenates.

    Science.gov (United States)

    Zhang, Jessie; Steinbach, Joe Henry

    2003-01-03

    Cytisine and nicotine bound to specific sites in homogenates prepared from HEK 293 cells which stably express human neuronal nicotinic alpha4 and beta2 subunits. The number of sites was the same for both ligands and nicotine was a full competitive inhibitor of cytisine binding. However, when binding was done to intact cells the number of binding sites per cell for nicotine was approximately 4-fold the number of sites for cytisine. Nicotine fully blocked cytisine binding, but cytisine only partially blocked nicotine binding to intact cells. When cells were permeabilized with saponin, the number of sites for nicotine was unchanged, while the number of sites for cytisine was increased, and cytisine was able to fully block nicotine binding. These data indicate that cytisine binds only to surface receptors on intact cells. The apparent affinity of cytisine for surface receptors (K(d)=0.8 nM) was not significantly different from that for receptors in the cell homogenate (0.3 nM).

  3. Lectin receptors on the plasma membrane of soybean cells. Binding and lateral diffusion of lectins.

    Science.gov (United States)

    Metcalf, T N; Wang, J L; Schubert, K R; Schindler, M

    1983-08-02

    Protoplasts prepared from suspension cultures of root cells of Glycine max (SB-1 cell line) bound soybean agglutinin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA). Binding studies carried out with 125I-labeled SBA, Con A, and WGA showed that these interactions were saturable and specific. Fluorescence microscopy demonstrated uniform membrane labeling. The mobility of the lectin-receptor complexes was measured by fluorescence redistribution after photobleaching. The diffusion constants (D) for SBA and Con A were 5 X 10(-11) and 7 X 10(-11) cm2/s, respectively. In contrast, WGA yielded a diffusion constant of 3 X 10(-10) cm2/s. Pretreatment of the protoplasts with either SBA or Con A resulted in a 6-fold reduction in the mobility of WGA (D congruent to 5 X 10(-11) cm2/s). The results suggest that the binding of SBA or Con A may lead to alterations of the soybean plasma membrane which, in turn, may restrict the mobility of other receptors.

  4. High affinity binding of /sup 125/I-labeled mouse interferon to a specific cell surface receptor. II. Analysis of binding properties

    Energy Technology Data Exchange (ETDEWEB)

    Aguet, M.; Blanchard, B.

    1981-12-01

    Direct ligand-binding studies with highly purified /sup 125/I-labeled virus-induced mouse interferon on mouse lymphoma L 1210 cells revealed a direct correlation of specific high-affinity binding with the biologic response to interferon. Neutralization of the antiviral effect by anti-interferon gamma globulin occurred at the same antibody concentration as the inhibition of specific binding. These results suggest that specific high-affinity binding of /sup 125/I-interferon occurred at a biologically functional interferon receptor. Competitive inhibition experiments using /sup 125/I- and /sup 127/I-labeled interferon provided strong evidence that the fraction of /sup 125/I-interferon inactivated upon labeling did not bind specifically. Scatchard analysis of the binding data yielded linear plots and thus suggested that interferon binds to homogeneous noncooperative receptor sites. In contrast to a characteristic property of several peptide hormone systems, binding of /sup 125/I-interferon to its specific receptor did not induce subsequent ligand degradation. At 37/sup o/ bound interferon was rapidly released in a biologically active form without evidence for molecular degradation. The expression of interferon receptors was not modified by treatment with interferon. Trypsin treatment of target cells and inhibition of protein synthesis abolished the specific binding of /sup 125/I-interferon. Three major molecular weight species of Newcastle disease virus-induced mouse C 243 cell interferon were isolated, separated, and identified as mouse ..cap alpha.. and ..beta.. interferons. These interferons were shown to inhibit competitively the specific binding of the highly purified labeled starting material thus providing evidence for a common receptor site for mouse interferon.

  5. Receptor-mediated binding and uptake of GnRH agonist and antagonist by pituitary cells

    Energy Technology Data Exchange (ETDEWEB)

    Jennes, L.; Stumpf, W.E.; Conn, P.M.

    1984-01-01

    The intracellular pathway of an enzyme resistant GnRH agonist (D- Lys6 -GnRH) conjugated to ferritin or to colloidal gold was followed in cultured pituitary cells. After an initial uniform distribution over the cell surface of gonadotropes, the electrondense marker was internalized, either individually or in small groups. After longer incubation times, the marker appeared in the lysosomal compartment and the Golgi apparatus, where it could be found in the vesicular as well as cisternal portion. In addition, the receptor-mediated endocytosis of the GnRH antagonist D-p-Glu1-D-Phe2-D-Trp3-D- Lys6 -GnRH was studied by light and electron microscopic autoradiography after 30 and 60 min of incubation to ensure uptake. At both time points, in in vitro as well as in vivo studies, silver grains were localized over cytoplasmic organelles of castration cells, including dilated endoplasmic reticulum, lysosomes, and clear vesicles. No consistent association with cell nuclei, mitochondria, or secretory vesicles could be observed. The results suggest that both agonist and antagonist are binding selectively to the plasma membrane of gonadotropes and subsequently are taken up via receptor-mediated endocytosis for degradation or possible action on synthetic processes.

  6. Androgen Receptor Localizes to Plasma Membrane by Binding to Caveolin-1 in Mouse Sertoli Cells.

    Science.gov (United States)

    Deng, Qiong; Wu, Yong; Zhang, Zeng; Wang, Yue; Li, Minghua; Liang, Hui; Gui, Yaoting

    2017-01-01

    The nonclassical androgen signaling pathway translates signals into alterations in cellular function within minutes, and this action is proposed to be mediated by an androgen receptor (AR) localized to the plasma membrane. This study was designed to determine the mechanism underlying the membrane association of androgen receptor in TM4 cells, a mouse Sertoli cell line. Western blot analysis indicated testosterone-induced AR translocation to the cell membrane. Data from coimmunoprecipitation indicated that AR is associated with caveolin-1, and testosterone enhanced this association. Knockdown of caveolin-1 by shRNA decreased the amount of AR localized to membrane fraction and prevented AR membrane trafficking after being exposed to testosterone at physiological concentration. The palmitoylation inhibitor 2-bromopalmitate decreased AR membrane localization in basal condition and completely blocked testosterone-induced AR translocation to membrane fraction. These data suggested that AR localized to membrane fraction by binding with caveolin-1 through palmitoylation of the cysteine residue. This study provided a new evidence for AR membrane localization and its application for clarifying the nonclassical signaling pathway of androgens.

  7. Androgen Receptor Localizes to Plasma Membrane by Binding to Caveolin-1 in Mouse Sertoli Cells

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    Qiong Deng

    2017-01-01

    Full Text Available The nonclassical androgen signaling pathway translates signals into alterations in cellular function within minutes, and this action is proposed to be mediated by an androgen receptor (AR localized to the plasma membrane. This study was designed to determine the mechanism underlying the membrane association of androgen receptor in TM4 cells, a mouse Sertoli cell line. Western blot analysis indicated testosterone-induced AR translocation to the cell membrane. Data from coimmunoprecipitation indicated that AR is associated with caveolin-1, and testosterone enhanced this association. Knockdown of caveolin-1 by shRNA decreased the amount of AR localized to membrane fraction and prevented AR membrane trafficking after being exposed to testosterone at physiological concentration. The palmitoylation inhibitor 2-bromopalmitate decreased AR membrane localization in basal condition and completely blocked testosterone-induced AR translocation to membrane fraction. These data suggested that AR localized to membrane fraction by binding with caveolin-1 through palmitoylation of the cysteine residue. This study provided a new evidence for AR membrane localization and its application for clarifying the nonclassical signaling pathway of androgens.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    G protein-coupled receptors (GPCRs) constitute a large class of seven transmembrane proteins, which bind selectively agonists or antagonists with important consequences for cellular signaling and function. Comprehension of the molecular details of ligand binding is important for the understanding...

  9. B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

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    Mattias N E Forsell

    2008-10-01

    Full Text Available The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3. Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4 rabbits with envelope glycoprotein (Env trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

  10. B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

    Science.gov (United States)

    Forsell, Mattias N E; Dey, Barna; Mörner, Andreas; Svehla, Krisha; O'dell, Sijy; Högerkorp, Carl-Magnus; Voss, Gerald; Thorstensson, Rigmor; Shaw, George M; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T

    2008-10-03

    The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

  11. Binding affinity to and dependence on some opioidsin Sf9 insect cells expressing human μ-opioid receptor

    Institute of Scientific and Technical Information of China (English)

    LIUZhong-Hua; HEYou; JINWen-Qiao; CHENXin-Jian; ZHANGHong-Ping; SHENQing-Xiang; CHIZhi-Qiang

    2003-01-01

    AIM: To investigate the receptor binding affinity and naloxone-precipitated cAMP overshoot of dihydroetorphine,fentanyl, heroin, and pethidine in Sf9 insect cells expressing human μ-opioid receptor (Sf9-μ cells). METHODS:Competitive binding assay of [3H]ohmefentanyl was used to reveal the affinity for μ-opioid receptor in Sf9-μ cells.[3H]cAMP RIA was used to determine cAMP level. Antinociceptive activity was evaluated using 55℃ mouse hotplate test. Naloxone-precipitated withdrawal jumping was used to reflect physical dependence in mice. RESULTS:All drugs displayed antinociceptive activity and produced physical dependence in mice. The Ki values ofdihydroetorphine, fentanyl, heroin, and pethidine in competitive binding assay were (0.85±0.20)nmol, (59.1±11.7)nmol, (0.36±0.13)μmol, and (12.2±3.8) μmol respectively. The binding affinities of these drugs for μ-opioidreceptor in Sf9-μ cells were paralleled to their antinociceptive activities in mice. After chronic pretreatment withthese drugs, naloxone induced cAMP withdrawal overshoot in Sf9-μ cells. The dependence index in Sf9-μ cellswas calculated as Ki value in competitive binding assay over ECs0 value in naloxone-precipitated cAMP assay, Thephysical dependence index in mice was calculated as antinociceptive ED50/withdrawal jumping cumulative EDs0.There was a good linear correlation between dependence index in Sf9-μ cells and physical dependence index inmice. CONCLUSION: The Sf9-μ cells could be used as a cell model to evaluate the receptor binding affinity andphysical dependent liability of analgesic agents.

  12. Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

    DEFF Research Database (Denmark)

    Mahony, Jennifer; Kot, Witold Piotr; Murphy, James;

    2013-01-01

    Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been impli...

  13. Investigation of the Relationship between Lactococcal Host Cell Wall Polysaccharide Genotype and 936 Phage Receptor Binding Protein Phylogeny

    DEFF Research Database (Denmark)

    Mahony, Jennifer; Kot, Witold Piotr; Murphy, James

    2013-01-01

    Comparative genomics of 11 lactococcal 936-type phages combined with host range analysis allowed subgrouping of these phage genomes, particularly with respect to their encoded receptor binding proteins. The so-called pellicle or cell wall polysaccharide of Lactococcus lactis, which has been impli...

  14. BINDING OF STEROIDS AND ENVIRONMENTAL CHEMICALS TO THE RAINBOW TROUT ANDROGEN RECEPTOR ALPHA EXPRESSED IN COS CELLS

    Science.gov (United States)

    Binding of Steroids and Environmental Chemicals to the Rainbow Trout Androgen Receptor Alpha Expressed in COS Cells. Mary C. Cardon, L. Earl Gray. Jr., Phillip C. Hartig and Vickie S. Wilson U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology...

  15. Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif.

    Science.gov (United States)

    Xu, Chenqi; Gagnon, Etienne; Call, Matthew E; Schnell, Jason R; Schwieters, Charles D; Carman, Christopher V; Chou, James J; Wucherpfennig, Kai W

    2008-11-14

    Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.

  16. 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.

  17. Machupo Virus Glycoprotein Determinants for Human Transferrin Receptor 1 Binding and Cell Entry

    Science.gov (United States)

    2011-07-01

    and form enveloped virions [1]. Seven arenaviruses cause viral hemorrhagic fever in humans: the Old World arenaviruses Lassa and ‘Lujo,’ and the New...hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to... fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular

  18. Evaluation of drug-muscarinic receptor affinities using cell membrane chromatography and radioligand binding assay in guinea pig jejunum membrane

    Institute of Scientific and Technical Information of China (English)

    Bing-xiang YUAN; Jin HOU; Lang-chong HE; Guang-de YANG

    2005-01-01

    Aim: To study if cell membrane chromatography (CMC) could reflect drug-receptor interaction and evaluate the affinity and competitive binding to muscarinic acetylcholine receptor (mAChR). Methods: The cell membrane stationary phase(CMSP) was prepared by immobilizing guinea pig jejunum cell membrane on the surface of a silica carrier, and was used for the rapid on-line chromatographic evaluation of ligand binding affinities to mAChR. The affinity to mAChR was also evaluated from radioligand binding assays (RBA) using the same jejunum membrane preparation. Results: The capacity factor (k') profiles in guinea pig jejunum CMSP were: (-)QNB (15.4)>(+)QNB (11.5)>atropine (5.35)>pirenzepine(5.26)>4-DAMP (4.45)>AF-DX 116 (4.18)>pilocarpine (3.93)>acetylcholine(1.31). These results compared with the affinity rank orders obtained from radioligand binding assays indicated that there wasa positive correlation (r2=0.8525, P<0.0001) between both data sets. Conclusion: The CMC method can be used to evaluate drug-receptor affinities for drug candidates.

  19. The Src homology 3 binding domain is required for lysophosphatidic acid 3 receptor-mediated cellular viability in melanoma cells.

    Science.gov (United States)

    Jia, Wei; Tran, Sterling K; Ruddick, Caitlin A; Murph, Mandi M

    2015-01-28

    The LPA3 receptor is a G protein-coupled receptor that binds extracellular lysophosphatidic acid and mediates intracellular signaling cascades. Although we previously reported that receptor inhibition using siRNA or chemical inhibition obliterates the viability of melanoma cells, the mechanism was unclear. Herein we hypothesized that amino acids comprising the Src homology 3 (SH3) ligand binding motif, R/K-X-X-V/P-X-X-P or (216)-KTNVLSP-(222), within the third intracellular loop of LPA3 were critical in mediating this outcome. Therefore, we performed site-directed mutagenesis of the lysine, valine and proline, replacing these amino acids with alanines, and evaluated the changes in viability, proliferation, ERK1/2 signaling and calcium in response to lysophosphatidic acid. Our results show that enforced LPA3 expression in SK-MEL-2 cells enhanced their resiliency by allowing these cells to oppose any loss of viability during growth in serum-free medium for up to 96 h, in contrast to parental SK-MEL-2 cells, which show a significant decline in viability. Similarly, site-directed alanine substitutions of valine and proline, V219A/P222A or 2aa-SK-MEL-2 cells, did not significantly alter viability, but adding a further alanine to replace the lysine, K216A/V219A/P222A or 3aa-SK-MEL-2 cells, obliterated this function. In addition, an inhibitor of the LPA3 receptor had no impact on the parental SK-MEL-2, 2aa-SK-MEL-2 or 3aa-SK-MEL-2 cells, but significantly reduced viability among wt-LPA3-SK-MEL-2 cells. Taken together, the data suggest that the SH3 ligand binding domain of LPA3 is required to mediate viability in melanoma cells.

  20. Selective permeabilization of cervical cancer cells to an ionic DNA-binding cytotoxin by activation of P2Y receptors.

    Science.gov (United States)

    Bukhari, Maurish; Deng, Han; Jones, Noelle; Towne, Zachary; Woodworth, Craig D; Samways, Damien S K

    2015-06-04

    Extracellular ATP is known to permeabilize certain cell types to polyatomic cations like YO-PRO1. Here, we report that extracellularly applied ATP stimulated rapid uptake and accumulation of an otherwise weakly membrane permeable fluorescent DNA-binding cytotoxin, Hoechst 33258, into cervical cancer cells. While ATP stimulated Hoechst 33258 uptake in 20-70% of cells from seven cervical cancer cell lines, it stimulated uptake in less than 8% of cervical epithelial cells obtained from the normal transformation zone and ectocervix tissue of 11 patients. ATP-evoked Hoechst 33258 uptake was independent of ionotropic P2X receptors, but dependent on activation of P2Y receptors. Thus, we show here that cervical cancer cells can be selectively induced to take up and accumulate an ionic cytotoxin by exposure to extracellular ATP.

  1. L1CAM Binds ErbB Receptors through Ig-Like Domains Coupling Cell Adhesion and Neuregulin Signalling

    Science.gov (United States)

    Grijota-Martinez, Carmen; Lakomá, Jarmila; Baars, Sigrid; Garcia-Alonso, Luis; Cabedo, Hugo

    2012-01-01

    During nervous system development different cell-to-cell communication mechanisms operate in parallel guiding migrating neurons and growing axons to generate complex arrays of neural circuits. How such a system works in coordination is not well understood. Cross-regulatory interactions between different signalling pathways and redundancy between them can increase precision and fidelity of guidance systems. Immunoglobulin superfamily proteins of the NCAM and L1 families couple specific substrate recognition and cell adhesion with the activation of receptor tyrosine kinases. Thus it has been shown that L1CAM-mediated cell adhesion promotes the activation of the EGFR (erbB1) from Drosophila to humans. Here we explore the specificity of the molecular interaction between L1CAM and the erbB receptor family. We show that L1CAM binds physically erbB receptors in both heterologous systems and the mammalian developing brain. Different Ig-like domains located in the extracellular part of L1CAM can support this interaction. Interestingly, binding of L1CAM to erbB enhances its response to neuregulins. During development this may synergize with the activation of erbB receptors through L1CAM homophilic interactions, conferring diffusible neuregulins specificity for cells or axons that interact with the substrate through L1CAM. PMID:22815787

  2. Estrogen receptor alpha binds to peroxisome proliferator-activated receptor response element and negatively interferes with peroxisome proliferator-activated receptor gamma signaling in breast cancer cells.

    Science.gov (United States)

    Bonofiglio, Daniela; Gabriele, Sabrina; Aquila, Saveria; Catalano, Stefania; Gentile, Mariaelena; Middea, Emilia; Giordano, Francesca; Andò, Sebastiano

    2005-09-01

    The molecular mechanisms involved in the repressive effects exerted by estrogen receptors (ER) on peroxisome proliferator-activated receptor (PPAR) gamma-mediated transcriptional activity remain to be elucidated. The aim of the present study was to provide new insight into the crosstalk between ERalpha and PPARgamma pathways in breast cancer cells. Using MCF7 and HeLa cells as model systems, we did transient transfections and electrophoretic mobility shift assay and chromatin immunoprecipitation studies to evaluate the ability of ERalpha to influence PPAR response element-mediated transcription. A possible direct interaction between ERalpha and PPARgamma was ascertained by co-immunoprecipitation assay, whereas their modulatory role in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway was evaluated by determining PI3K activity and AKT phosphorylation. As a biological counterpart, we investigated the growth response to the cognate ligands of both receptors in hormone-dependent MCF7 breast cancer cells. Our data show for the first time that ERalpha binds to PPAR response element and represses its transactivation. Moreover, we have documented the physical and functional interactions of ERalpha and PPARgamma, which also involve the p85 regulatory subunit of PI3K. Interestingly, ERalpha and PPARgamma pathways have an opposite effect on the regulation of the PI3K/AKT transduction cascade, explaining, at least in part, the divergent response exerted by the cognate ligands 17beta-estradiol and BRL49653 on MCF7 cell proliferation. ERalpha physically associates with PPARgamma and functionally interferes with PPARgamma signaling. This crosstalk could be taken into account in setting new pharmacologic strategies for breast cancer disease.

  3. Human adenovirus 52 uses sialic acid-containing glycoproteins and the coxsackie and adenovirus receptor for binding to target cells.

    Directory of Open Access Journals (Sweden)

    Annasara Lenman

    2015-02-01

    Full Text Available Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR protein. Human adenovirus type 52 (HAdV-52 is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK binds to CAR, and the knob domain of the short fiber (52SFK binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.

  4. Structure-activity relationship of imidazothiadiazole analogs for the binding to the ecdysone receptor of insect cells.

    Science.gov (United States)

    Yokoi, Taiyo; Minami, Saki; Nakagawa, Yoshiaki; Miyagawa, Hisashi

    2015-05-01

    Diacylhydrazines are the first non-steroidal ecdysone agonists, and five compounds are used as insecticides in agriculture. After the discovery of diacylhydrazine-type compounds, numerous non-steroidal structures were reported as ecdysone agonists. Among various ecdysone agonists, imidazothiadiazoles are reported to be very potent in vitro; however, the experimental detail for the structure identification and bioassays are not stated in the paper (Holmwood and Schindler, Bioorg. Med. Chem. 17, 4064-4070, 2009). In our present study, we synthesized 18 imidazothiadiazole-type compounds and confirmed the chemical structures by spectrometric analyses. The binding activity of the synthesized compounds to the ecdysone receptor was evaluated in terms of the concentration required for 50% inhibition of [(3)H]ponasterone A incorporation [IC50 (M)] into lepidopteran (Sf-9), coleopteran (BCRL-Lepd-SL1), and dipteran (NIAS-AeAl2) cells. 6-(2-Chlorophenyl)-2-(trifluoromethyl)imidazo[2,1-b] [1,3,4]-thiadiazol-5-yl)acrylamide analogs with CONHR (secondary amide) were very potent against Sf-9 cells, but further alkylation (tertiary amide: CONR2) decreased the activity dramatically. Additionally, a primary amide analog (CONH2) was inactive. The activity also decreased 150-fold by the saturation of olefin region of the acrylamide moiety. In addition, various substituents were introduced at the 2-position of the imidazothiadiazole ring to disclose the physicochemical properties of the substituents which are important for receptor binding. The activity increased by 7500-fold with the introduction of the CF2CF2CF3 group compared to the unsubstituted compound against Sf-9 cells. Quantitative structure-activity relationship analysis for these substituents indicated that hydrophobic and electron-withdrawing groups were favorable for binding. Some of the compounds with strong receptor binding activity showed good larvicidal activity against Spodoptera litura. In contrast, the binding

  5. The retinol esterifying enzyme LRAT supports cell signaling by retinol-binding protein and its receptor STRA6.

    Science.gov (United States)

    Marwarha, Gurdeep; Berry, Daniel C; Croniger, Colleen M; Noy, Noa

    2014-01-01

    Vitamin A, retinol, circulates in blood bound to retinol-binding protein (RBP). At some tissues, holo-RBP is recognized by a plasma membrane receptor termed STRA6, which serves a dual role: it mediates transport of retinol from RBP into cells, and it functions as a cytokine receptor that, on binding holo-RBP, activates JAK2/STAT5 signaling. As STAT target genes include SOCS3, an inhibitor of insulin receptor, holo-RBP suppresses insulin responses in STRA6-expressing cells. We have shown previously that the two functions of STRA6 are interdependent. These observations suggest factors that regulate STRA6-mediated retinol transport may also control STRA6-mediated cell signaling. One such factor is retinol metabolism, which enables cellular uptake of retinol by maintaining an inward-directed concentration gradient. We show here that lecithin:retinol acyl transferase (LRAT), which catalyzes esterification of retinol to its storage species retinyl esters, is necessary for activation of the STRA6/JAK2/STAT5 cascade by holo-RBP. In accordance, LRAT-null mice are protected from holo-RBP-induced suppression of insulin responses. Hence, STRA6 signaling, which requires STRA6-mediated retinol transport, is supported by LRAT-catalyzed retinol metabolism. The observations demonstrate that STRA6 regulates key cellular processes by coupling circulating holo-RBP levels and intracellular retinol metabolism to cell signaling.

  6. Urokinase plasminogen activator cleaves its cell surface receptor releasing the ligand-binding domain

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Rønne, E; Solberg, H

    1992-01-01

    -domain form, uPAR(2+3), lacking ligand-binding domain 1. Trypsin treatment showed that both variants are present on the outside of the cells. Addition to the culture medium of an anticatalytic monoclonal antibody to uPA inhibited the formation of the uPAR(2+3), indicating that uPA is involved in its...

  7. Short-term desensitization of muscarinic cholinergic receptors in mouse neuroblastoma cells: selective loss of agonist low-affinity and pirenzepine high-affinity binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Cioffi, C.L.; el-Fakahany, E.E.

    1986-09-01

    The effects of brief incubation with carbamylcholine on subsequent binding of (/sup 3/H)N-methylscopolamine were investigated in mouse neuroblastoma cells (clone N1E-115). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one which is readily susceptible to regulation by receptor agonists, whereas the other is resistant in this regard. In control cells, both pirenzepine and carbamylcholine interacted with high- and low-affinity subsets of muscarinic receptors. Computer-assisted analysis of the competition between pirenzepine and carbamylcholine with (/sup 3/H)N-methylscopolamine showed that the receptor sites remaining upon desensitization are composed mainly of pirenzepine low-affinity and agonist high-affinity binding sites. Furthermore, there was an excellent correlation between the ability of various muscarinic receptor agonists to induce a decrease in consequent (/sup 3/H)N-methylscopolamine binding and their efficacy in stimulating cyclic GMP synthesis in these cells. Thus, only the agonists that are known to recognize the receptor's low-affinity conformation in order to elicit increases in cyclic GMP levels were capable of diminishing ligand binding. Taken together, our present results suggest that the receptor population that is sensitive to regulation by agonists includes both the pirenzepine high-affinity and the agonist low-affinity receptor binding states. In addition, the sensitivity of these receptor subsets to rapid regulation by agonists further implicates their involvement in desensitization of muscarinic receptor-mediated cyclic GMP formation.

  8. An overview of nomegestrol acetate selective receptor binding and lack of estrogenic action on hormone-dependent cancer cells.

    Science.gov (United States)

    Shields-Botella, J; Duc, I; Duranti, E; Puccio, F; Bonnet, P; Delansorne, R; Paris, J

    2003-11-01

    The specific pharmacological profile of the 19-norprogestin nomegestrol acetate (NOMAC) is, at least in part, defined by its pattern of binding affinities to the different steroid hormone receptors. In the present study, its affinity to the progesterone receptor (PgR), the androgen receptor (AR) and the estrogen receptor (ER) was re-evaluated and compared to those obtained for progesterone (P) and several progestins. The characteristics of binding to the PgR in rat uterus were determined and Ki were found to be roughly similar with 22.8 and 34.3 nM for NOMAC and P, respectively. The binding characteristics of 3H-NOMAC were also determined and compared to that of 3H-ORG2058 with Kd of 5 and 0.6 nM, respectively for rat uterus and 4 and 3 nM, respectively for human T47-D cells. Structure-affinity and -activity relationships were studied on a variety of compounds related to NOMAC in order to assess its specificity as a progestin. The effects of NOMAC on the binding of androgen to the AR were investigated, using rat ventral prostate as target model. Contrary to what was observed for MPA, the RBA of NOMAC was found to decline with time, showing anti-androgenic rather than androgenic potential, a result that was confirmed in vivo. Regarding the ER, since none of the progestins were able to compete with estrogen for binding in rat uterus as well as in Ishikawa cells, the induction of alkaline phosphatase activity (APase) was used as an estrogen-specific response. It confirmed the intrinsic estrogenicity of progestins derived from 19-nor-testosterone (19NT), norethisterone acetate (NETA), levonorgestrel (LNG) or norgestimate (NGM) and others. In contrast, all P and 19-norP derivatives remained inactive. Finally, to complete this overview of NOMAC at the sex steroid receptor levels, the lack of estrogenic or estrogenic-like activity was checked out in different in vitro models. Data from this study have demonstrated that NOMAC is a progestin that has greater steroid

  9. HIV gp120 binds to mannose receptor on vaginal epithelial cells and induces production of matrix metalloproteinases.

    Directory of Open Access Journals (Sweden)

    Sashaina E Fanibunda

    Full Text Available BACKGROUND: During sexual transmission of HIV in women, the virus breaches the multi-layered CD4 negative stratified squamous epithelial barrier of the vagina, to infect the sub-epithelial CD4 positive immune cells. However the mechanisms by which HIV gains entry into the sub-epithelial zone is hitherto unknown. We have previously reported human mannose receptor (hMR as a CD4 independent receptor playing a role in HIV transmission on human spermatozoa. The current study was undertaken to investigate the expression of hMR in vaginal epithelial cells, its HIV gp120 binding potential, affinity constants and the induction of matrix metalloproteinases (MMPs downstream of HIV gp120 binding to hMR. PRINCIPAL FINDINGS: Human vaginal epithelial cells and the immortalized vaginal epithelial cell line Vk2/E6E7 were used in this study. hMR mRNA and protein were expressed in vaginal epithelial cells and cell line, with a molecular weight of 155 kDa. HIV gp120 bound to vaginal proteins with high affinity, (Kd = 1.2±0.2 nM for vaginal cells, 1.4±0.2 nM for cell line and the hMR antagonist mannan dose dependently inhibited this binding. Both HIV gp120 binding and hMR exhibited identical patterns of localization in the epithelial cells by immunofluorescence. HIV gp120 bound to immunopurified hMR and affinity constants were 2.9±0.4 nM and 3.2±0.6 nM for vaginal cells and Vk2/E6E7 cell line respectively. HIV gp120 induced an increase in MMP-9 mRNA expression and activity by zymography, which could be inhibited by an anti-hMR antibody. CONCLUSION: hMR expressed by vaginal epithelial cells has high affinity for HIV gp120 and this binding induces production of MMPs. We propose that the induction of MMPs in response to HIV gp120 may lead to degradation of tight junction proteins and the extracellular matrix proteins in the vaginal epithelium and basement membrane, leading to weakening of the epithelial barrier; thereby facilitating transport of HIV across the

  10. Regulation of T cell Receptor Activation by Dynamic Membrane Binding of the CD3ε Cytoplasmic Tyrosine-Based Motif

    Science.gov (United States)

    Xu, Chenqi; Gagnon, Etienne; Call, Matthew E.; Schnell, Jason R.; Schwieters, Charles D.; Carman, Christopher V.; Chou, James J.; Wucherpfennig, Kai W.

    2008-01-01

    Summary Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live cell imaging studies showed a close interaction of the CD3ε cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer (FRET) between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3ε residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance (NMR) structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3ε ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation. PMID:19013279

  11. Soluble T cell receptor Vβ domains engineered for high-affinity binding to staphylococcal or streptococcal superantigens.

    Science.gov (United States)

    Sharma, Preeti; Wang, Ningyan; Kranz, David M

    2014-01-28

    Staphylococcus aureus and group A Streptococcus secrete a collection of toxins called superantigens (SAgs), so-called because they stimulate a large fraction of an individual's T cells. One consequence of this hyperactivity is massive cytokine release leading to severe tissue inflammation and, in some cases, systemic organ failure and death. The molecular basis of action involves the binding of the SAg to both a T cell receptor (TCR) on a T cell and a class II product of the major histocompatibility complex (MHC) on an antigen presenting cell. This cross-linking leads to aggregation of the TCR complex and signaling. A common feature of SAgs is that they bind with relatively low affinity to the variable region (V) of the beta chain of the TCR. Despite this low affinity binding, SAgs are very potent, as each T cell requires only a small fraction of their receptors to be bound in order to trigger cytokine release. To develop high-affinity agents that could neutralize the activity of SAgs, and facilitate the development of detection assays, soluble forms of the Vβ regions have been engineered to affinities that are up to 3 million-fold higher for the SAg. Over the past decade, six different Vβ regions against SAgs from S. aureus (SEA, SEB, SEC3, TSST-1) or S. pyogenes (SpeA and SpeC) have been engineered for high-affinity using yeast display and directed evolution. Here we review the engineering of these high-affinity Vβ proteins, structural features of the six different SAgs and the Vβ proteins, and the specific properties of the engineered Vβ regions that confer high-affinity and specificity for their SAg ligands.

  12. Soluble T Cell Receptor Vβ Domains Engineered for High-Affinity Binding to Staphylococcal or Streptococcal Superantigens

    Directory of Open Access Journals (Sweden)

    Preeti Sharma

    2014-01-01

    Full Text Available Staphylococcus aureus and group A Streptococcus secrete a collection of toxins called superantigens (SAgs, so-called because they stimulate a large fraction of an individual’s T cells. One consequence of this hyperactivity is massive cytokine release leading to severe tissue inflammation and, in some cases, systemic organ failure and death. The molecular basis of action involves the binding of the SAg to both a T cell receptor (TCR on a T cell and a class II product of the major histocompatibility complex (MHC on an antigen presenting cell. This cross-linking leads to aggregation of the TCR complex and signaling. A common feature of SAgs is that they bind with relatively low affinity to the variable region (V of the beta chain of the TCR. Despite this low affinity binding, SAgs are very potent, as each T cell requires only a small fraction of their receptors to be bound in order to trigger cytokine release. To develop high-affinity agents that could neutralize the activity of SAgs, and facilitate the development of detection assays, soluble forms of the Vβ regions have been engineered to affinities that are up to 3 million-fold higher for the SAg. Over the past decade, six different Vβ regions against SAgs from S. aureus (SEA, SEB, SEC3, TSST-1 or S. pyogenes (SpeA and SpeC have been engineered for high-affinity using yeast display and directed evolution. Here we review the engineering of these high-affinity Vβ proteins, structural features of the six different SAgs and the Vβ proteins, and the specific properties of the engineered Vβ regions that confer high-affinity and specificity for their SAg ligands.

  13. Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (. mu. ) and enkephalin (delta) binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Kazmi, S.M.I.; Mishra, R.K.

    1986-06-13

    Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of ..mu.. and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of (/sup 3/H)-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin (DADLE) in the presence of 10/sup -5/M D-Pro/sup 4/-morphiceptin (to block the ..mu.. receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of (/sup 3/H)-dihydromorphine, together with the higher potency of morphine analogues to displace (/sup 3/H)-naloxone binding established the presence of ..mu.. sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of (/sup 3/H)-DADLE binding.

  14. Characterization of Plasminogen Binding to NB4 Promyelocytic Cells Using Monoclonal Antibodies against Receptor-Induced Binding Sites in Cell-Bound Plasminogen

    Directory of Open Access Journals (Sweden)

    Mercè Jardí

    2012-01-01

    Full Text Available The NB4 promyelocytic cell line exhibits many of the characteristics of acute promyelocytic leukemia blast cells, including the translocation (15 : 17 that fuses the PML gene on chromosome 15 to the RARα gene on chromosome 17. These cells have a very high fibrinolytic capacity. In addition to a high secretion of urokinase, NB4 cells exhibit a 10-fold higher plasminogen binding capacity compared with other leukemic cell lines. When tissue-type plasminogen activator was added to acid-treated cells, plasmin generation was 20–26-fold higher than that generated by U937 cells or peripheral blood neutrophils, respectively. We found that plasminogen bound to these cells can be detected by fluorescence-activated cell sorting using an antiplasminogen monoclonal antibody that specifically reacts with this antigen when it is bound to cell surfaces. All-trans retinoid acid treatment of NB4 cells markedly decreased the binding of this monoclonal antibody. This cell line constitutes a unique model to explore plasminogen binding and activation on cell surfaces that can be modulated by all-trans retinoid acid treatment.

  15. Genome-wide progesterone receptor binding: cell type-specific and shared mechanisms in T47D breast cancer cells and primary leiomyoma cells.

    Directory of Open Access Journals (Sweden)

    Ping Yin

    Full Text Available BACKGROUND: Progesterone, via its nuclear receptor (PR, exerts an overall tumorigenic effect on both uterine fibroid (leiomyoma and breast cancer tissues, whereas the antiprogestin RU486 inhibits growth of these tissues through an unknown mechanism. Here, we determined the interaction between common or cell-specific genome-wide binding sites of PR and mRNA expression in RU486-treated uterine leiomyoma and breast cancer cells. PRINCIPAL FINDINGS: ChIP-sequencing revealed 31,457 and 7,034 PR-binding sites in breast cancer and uterine leiomyoma cells, respectively; 1,035 sites overlapped in both cell types. Based on the chromatin-PR interaction in both cell types, we statistically refined the consensus progesterone response element to G•ACA• • •TGT•C. We identified two striking differences between uterine leiomyoma and breast cancer cells. First, the cis-regulatory elements for HSF, TEF-1, and C/EBPα and β were statistically enriched at genomic RU486/PR-targets in uterine leiomyoma, whereas E2F, FOXO1, FOXA1, and FOXF sites were preferentially enriched in breast cancer cells. Second, 51.5% of RU486-regulated genes in breast cancer cells but only 6.6% of RU486-regulated genes in uterine leiomyoma cells contained a PR-binding site within 5 kb from their transcription start sites (TSSs, whereas 75.4% of RU486-regulated genes contained a PR-binding site farther than 50 kb from their TSSs in uterine leiomyoma cells. RU486 regulated only seven mRNAs in both cell types. Among these, adipophilin (PLIN2, a pro-differentiation gene, was induced via RU486 and PR via the same regulatory region in both cell types. CONCLUSIONS: Our studies have identified molecular components in a RU486/PR-controlled gene network involved in the regulation of cell growth, cell migration, and extracellular matrix function. Tissue-specific and common patterns of genome-wide PR binding and gene regulation may determine the therapeutic effects of antiprogestins in

  16. Isolation and characterization of a cyanobacterium-binding protein and its cell wall receptor in the lichen Peltigera canina.

    Science.gov (United States)

    Díaz, Eva-María; Sacristán, Mara; Legaz, María-Estrella; Vicente, Carlos

    2009-07-01

    Peltigera canina, a cyanolichen containing Nostoc as cyanobiont, produces and secretes arginase to a medium containing arginine. Secreted arginase acts as a lectin by binding to the surface of Nostoc cells through a specific receptor which develops urease activity. The enzyme urease has been located in the cell wall of recently isolated cyanobionts. Cytochemical detection of urease is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO(2) evolved from urea hydrolysis in the presence of cobalt chloride. This urease has been pre-purified by affinity chromatography on a bead of active agarose to which arginase was attached. Urease was eluted from the beads by 50 mM alpha-D-galactose. The experimentally probed fact that a fungal lectin developing subsidiary arginase activity acts as a recognition factor of compatible algal cells in chlorolichens can now been expanded to cyanolichens.

  17. Characterization of a Putative Receptor Binding Surface on Skint-1, a Critical Determinant of Dendritic Epidermal T Cell Selection*

    Science.gov (United States)

    Salim, Mahboob; Knowles, Timothy J.; Hart, Rosie; Mohammed, Fiyaz; Woodward, Martin J.; Willcox, Carrie R.; Overduin, Michael; Hayday, Adrian C.; Willcox, Benjamin E.

    2016-01-01

    Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection. PMID:26917727

  18. CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

    DEFF Research Database (Denmark)

    1995-01-01

    Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

  19. [Selective regulation of peroxisome proliferator-activated receptors on fatty acid binding protein-4 in human syncytiotrophoblast cells].

    Science.gov (United States)

    Li, Yuan; Gu, Hang

    2012-10-01

    To observe the selective regulation of peroxisome proliferator-activated receptors (PPAR) on fatty acid binding protein-4 (FABP4) in human syncytiotrophoblasts. Cultivate normal human syncytiotrophoblast cells, and put in the specific antagonists and agonists of PPAR each subtypes receptors, then observe the different expression of FABP4 mRNA and protein. Pretreated the human syncytiotrophoblast cells with the agonists (GW7647, GW0742) and antagonists (GW6471, GSK0660) of PPARα and PPARβ receptors, the expression of the FABP4 was not significantly change (P > 0.05). However pretreated with PPAR γ agonists (rosiglitazone, 1×10(-9), 1×10(-8), 1×10(-7) and 1×10(-6) mol/L), the expression of FABP4 mRNA and protein could be dose dependent-promoted significantly (mRNA: 1.27 ± 0.12, 1.45 ± 0.14, 1.57 ± 0.14, 1.72 ± 0.12, protein:1.10 ± 0.08, 1.37 ± 0.09, 1.60 ± 0.13, 1.79 ± 0.14; P PPARγ regulated the expression of FABP4 mRNA and protein selectively. And the regulation will not be influenced by the other two PPAR subtypes.

  20. Neural cell adhesion molecule-180-mediated homophilic binding induces epidermal growth factor receptor (EGFR) down-regulation and uncouples the inhibitory function of EGFR in neurite outgrowth

    DEFF Research Database (Denmark)

    Povlsen, Gro Klitgaard; Berezin, Vladimir; Bock, Elisabeth

    2008-01-01

    The neural cell adhesion molecule (NCAM) plays important roles in neuronal development, regeneration, and synaptic plasticity. NCAM homophilic binding mediates cell adhesion and induces intracellular signals, in which the fibroblast growth factor receptor plays a prominent role. Recent studies...... not require NCAM-mediated fibroblast growth factor receptor activation....... on axon guidance in Drosophila suggest that NCAM also regulates the epidermal growth factor receptor (EGFR) (Molecular and Cellular Neuroscience, 28, 2005, 141). A possible interaction between NCAM and EGFR in mammalian cells has not been investigated. The present study demonstrates for the first time...

  1. Asbestos fibre length-dependent detachment injury to alveolar epithelial cells in vitro: role of a fibronectin-binding receptor.

    Science.gov (United States)

    Donaldson, K.; Miller, B. G.; Sara, E.; Slight, J.; Brown, R. C.

    1993-01-01

    A short and a long fibre sample of amosite asbestos were tested for their effects on cells of the human Type 2 alveolar epithelial cell-line A549 in vitro. The long amosite sample was found to cause a rapid detachment of the epithelial cells live from their substratum. At the highest dose, on average 28% of the cells present were detached in this way. Studies on the mechanism of the detachment injury showed that it did not involve oxidants since it was not ameliorated by scavengers of active oxygen species. Neither was the effect reduced by treatment of the fibres with the iron chelator Desferal. Treatments reported to increase the interaction between fibres and cells, serum and poly-L-lysine, did not influence the detachment injury, nor did lung lining fluid. Conversely, the fibronectin tripeptide RGD alone could cause detachment which suggested that a fibronectin-binding integrin was involved. This receptor could be reduced in activity by long fibre exposure, leading to detachment. The detaching effect of fibre could be mimicked by the protein kinase C activator PMA, and so the second messenger system of the cell could also be involved. This type of injury could be important in the pathology associated with exposure to long fibres. PMID:8392859

  2. DC-SIGN:Binding receptor for HCV?

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hua Feng; Quan-Chu Wang; Qing-He Nie; Zhan-Sheng Jia; Yong-Xin Zhou

    2004-01-01

    DC-SIGN, a dendritic Cell-specific adhesion receptor and a type Ⅱ transmembrane mannose-binding C-type lectin, is very important in the function of DC, both in mediating naive T cell interactions through ICAM-3 and as a rolling receptor that mediates the DC-specific ICAM-2-dependent migration processes. It can be used by viral and bacterial pathogens including Human Immunodeficiency Virus (HIV), HCV, Ebola Virus, CMV and Mycobacterium tuberculosis to facilitate infection. Both DC-SIGN and DC-SIGNR can act either in cis,by concentrating virus on target cells, or in trans, by transmission of bound virus to a target cell expressing appropropriate entry receptors. Recent work showed that DC-SIGN are highaffinity binding receptors for HCV. Besides playing a role in entry into DC, HCV E2 interaction with DC-SIGN might also be detrimental for the interaction of DC with T cells during antigen presentation. The clinical strategies that target DCSIGN may be successful in restricting HCV dissemination and pathogenesis as well as directing the migration of DCs to manipulate appropriate immune responses in autoimmunity and tumorigenic situations.

  3. Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory.

    Science.gov (United States)

    Weikl, Thomas R; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

    2016-09-02

    The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant [Formula: see text] and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between [Formula: see text] and the binding constant [Formula: see text] of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D).

  4. T-cell receptors binding orientation over peptide/MHC class I is driven by long-range interactions.

    Directory of Open Access Journals (Sweden)

    Mathias Ferber

    Full Text Available Crystallographic data about T-Cell Receptor - peptide - major histocompatibility complex class I (TCRpMHC interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes.

  5. Antitumor Responses Stimulated by Dendritic Cells Are Improved by Triiodothyronine Binding to the Thyroid Hormone Receptor β.

    Science.gov (United States)

    Alamino, Vanina A; Mascanfroni, Iván D; Montesinos, María M; Gigena, Nicolás; Donadio, Ana C; Blidner, Ada G; Milotich, Sonia I; Cheng, Sheue-Yann; Masini-Repiso, Ana M; Rabinovich, Gabriel A; Pellizas, Claudia G

    2015-04-01

    Bidirectional cross-talk between the neuroendocrine and immune systems orchestrates immune responses in both physiologic and pathologic settings. In this study, we provide in vivo evidence of a critical role for the thyroid hormone triiodothyronine (T3) in controlling the maturation and antitumor functions of dendritic cells (DC). We used a thyroid hormone receptor (TR) β mutant mouse (TRβPV) to establish the relevance of the T3-TRβ system in vivo. In this model, TRβ signaling endowed DCs with the ability to stimulate antigen-specific cytotoxic T-cell responses during tumor development. T3 binding to TRβ increased DC viability and augmented DC migration to lymph nodes. Moreover, T3 stimulated the ability of DCs to cross-present antigens and to stimulate cytotoxic T-cell responses. In a B16-OVA mouse model of melanoma, vaccination with T3-stimulated DCs inhibited tumor growth and prolonged host survival, in part by promoting the generation of IFNγ-producing CD8(+) T cells. Overall, our results establish an adjuvant effect of T3-TRβ signaling in DCs, suggesting an immediately translatable method to empower DC vaccination approaches for cancer immunotherapy.

  6. Peroxisome Proliferator-Activated Receptorα Agonists Differentially Regulate Inhibitor of DNA Binding Expression in Rodents and Human Cells

    Directory of Open Access Journals (Sweden)

    María del Carmen González

    2012-01-01

    Full Text Available Inhibitor of DNA binding (Id2 is a helix-loop-helix (HLH transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY. WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2. MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V, the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.

  7. Peroxisome proliferator-activated receptorα agonists differentially regulate inhibitor of DNA binding expression in rodents and human cells.

    Science.gov (United States)

    González, María Del Carmen; Corton, J Christopher; Acero, Nuria; Muñoz-Mingarro, Dolores; Quirós, Yolanda; Alvarez-Millán, Juan José; Herrera, Emilio; Bocos, Carlos

    2012-01-01

    Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.

  8. Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

    Energy Technology Data Exchange (ETDEWEB)

    Bencherif, M.; Lukas, R.J. (Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (USA))

    1991-06-01

    Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.

  9. The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4+ cells

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2012-02-01

    Full Text Available -1 Virology 423 (2012) 175?186 The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4+ cells Kabamba B. Alexandre a, Elin S. Gray a, Hazel Mufhandu b, James B. McMahon c, Ereck Chakauya b...

  10. Formyl peptide receptor chimeras define domains involved in ligand binding.

    Science.gov (United States)

    Perez, H D; Holmes, R; Vilander, L R; Adams, R R; Manzana, W; Jolley, D; Andrews, W H

    1993-02-05

    We have begun to study the structural requirements for the binding of formyl peptides to their specific receptors. As an initial approach, we constructed C5a-formyl peptide receptor chimeras. Unique (and identical) restriction sites were introduced within the transmembrane domains of these receptors that allowed for the exchange of specific areas. Four types of chimeric receptors were generated. 1) The C5a receptor was progressively substituted by the formyl peptide receptor. 2) The formyl peptide receptor was progressively substituted by the C5a receptor. 3) Specific domains of the C5a receptor were substituted by the corresponding domain of the formyl peptide receptor. 4) Specific domains of the formyl peptide receptor were replaced by the same corresponding domain of the C5a receptor. Wild type and chimeric receptors were transfected into COS 7 cells and their ability to bind formyl peptide determined, taking into account efficiency of transfection and expression of chimeric protein. Based on these results, a ligand binding model is presented in which the second, third, and fourth extracellular (and/or their transmembrane) domains together with the first transmembrane domain form a ligand binding pocket for formyl peptides. It is proposed that the amino-terminal domain plays a role by presumably providing a "lid" to the pocket. The carboxyl-terminal cytoplasmic tail appears to modulate ligand binding by regulating receptor affinity.

  11. Residues essential for Panton-Valentine leukocidin S component binding to its cell receptor suggest both plasticity and adaptability in its interaction surface.

    Directory of Open Access Journals (Sweden)

    Benoit-Joseph Laventie

    Full Text Available Panton-Valentine leukocidin (PVL, a bicomponent staphylococcal leukotoxin, is involved in the poor prognosis of necrotizing pneumonia. The present study aimed to elucidate the binding mechanism of PVL and in particular its cell-binding domain. The class S component of PVL, LukS-PV, is known to ensure cell targeting and exhibits the highest affinity for the neutrophil membrane (Kd∼10(-10 M compared to the class F component of PVL, LukF-PV (Kd∼10(-9 M. Alanine scanning mutagenesis was used to identify the residues involved in LukS-PV binding to the neutrophil surface. Nineteen single alanine mutations were performed in the rim domain previously described as implicated in cell membrane interactions. Positions were chosen in order to replace polar or exposed charged residues and according to conservation between leukotoxin class S components. Characterization studies enabled to identify a cluster of residues essential for LukS-PV binding, localized on two loops of the rim domain. The mutations R73A, Y184A, T244A, H245A and Y250A led to dramatically reduced binding affinities for both human leukocytes and undifferentiated U937 cells expressing the C5a receptor. The three-dimensional structure of five of the mutants was determined using X-ray crystallography. Structure analysis identified residues Y184 and Y250 as crucial in providing structural flexibility in the receptor-binding domain of LukS-PV.

  12. Porphyrin analogues as novel antagonists of fibroblast growth factor and vascular endothelial growth factor receptor binding that inhibit endothelial cell proliferation, tumor progression, and metastasis.

    Science.gov (United States)

    Aviezer, D; Cotton, S; David, M; Segev, A; Khaselev, N; Galili, N; Gross, Z; Yayon, A

    2000-06-01

    Fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF) play a pivotal role in the multistep pathway of tumor progression, metastasis, and angiogenesis. We have identified a porphyrin analogue, 5,10,15,20-tetrakis(methyl-4-pyridyl)-21H,23H-porphine-tetra -p-tosylate salt (TMPP), as a potent inhibitor of FGF2 and VEGF receptor binding and activation. TMPP demonstrated potent inhibition of binding of soluble FGF receptor 1 (FGFR1) to FGF2 immobilized on heparin at submicromolar concentrations. TMPP inhibits binding of radiolabeled FGF2 to FGFR in a cell-free system as well as to cells genetically engineered to express FGFR1. Furthermore, TMPP also inhibits the binding of VEGF to its tyrosine kinase receptor in a dose-dependent manner. In an in vitro angiogenic assay measuring the extent of endothelial cell growth, tube formation, and sprouting, TMPP dramatically reduced the extent of the FGF2-induced endothelial cell outgrowth and differentiation. In a Lewis lung carcinoma model, mice receiving TMPP showed a marked inhibition of both primary tumor progression and lung metastases development, with nearly total inhibition of the metastatic phenotype upon alternate daily injections of TMPP at 25 microg/g of body mass. Finally, novel meso-pyridylium-substituted, nonsymmetric porphyrins, as well as a novel corrole-based derivative, with >50-fold increase in activity in vitro, had a significantly improved efficacy in blocking tumor progression and metastasis in vivo.

  13. The cell surface expression of SAP-binding receptor CD229 is regulated via its interaction with clathrin-associated adaptor complex 2 (AP-2).

    Science.gov (United States)

    Del Valle, Juana M; Engel, Pablo; Martín, Margarita

    2003-05-09

    CD229 (Ly9) is a cell surface receptor selectively expressed on T and B lymphocytes, and it belongs to the CD150 receptor family. Like other receptors of this family, CD229 interacts with SAP/SH2D1a protein, mutation of which is responsible for the fatal X-linked lymphoproliferative disease. Receptors of the CD150 family function as costimulatory molecules, regulating cytokine production and cytotoxicity. Thus, their signaling and regulation in lymphocytes may be critical to an understanding of the pathogenesis of the X-linked lymphoproliferative disease. Here we show that CD229 interacts with the mu(2) chain of the AP-2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD229 was the only member of the CD150 family associated with AP-2. We also show that the mu(2) chain interacts with the Y(470)EKL motif of CD229. The integrity of this site was necessary for CD229 internalization, but it was not involved in SAP recruitment. Moreover, CD229 binds to the AP-2 complex in T and B cell lines, and it is internalized rapidly from the cell surface on T cells after antibody ligation. In contrast, cross-linking of CD229 receptors with intact antibody inhibited CD229 internalization on B cells. However, when F(ab')(2) antibodies were used, CD229 internalization was similar on T and B cells, suggesting that Fcgamma receptors control CD229 cell surface expression. Furthermore, CD229 was regulated by T cell receptor and B cell receptor signaling because coligation with antibodies against anti-CD3 and anti-IgM increased the rate of CD229 endocytosis. These data suggest that CD229 cell surface expression on lymphocytes surface is strongly and differentially regulated within the CD150 family members.

  14. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    Energy Technology Data Exchange (ETDEWEB)

    Serck-Hanssen, G.; Soevik, O.

    1987-12-28

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of /sup 125/I-insulin was carried out at 15/sup 0/C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table.

  15. Identification of a prostacyclin receptor coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mouse mastocytoma P-815 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, H.; Negishi, M.; Ichikawa, A. (Kyoto Univ. (Japan))

    1990-11-01

    A stable analogue of prostacyclin, iloprost, specifically bound to 30,000 x g pellet (the membrane fraction) prepared from mouse mastocytoma P-815 cells. The binding was dependent on time, temperature and pH, and absolutely required a divalent cation. The equilibrium dissociation constant and the maximal concentration of the binding site as determined by Scatchard plot analysis were 10.4 nM and 1.12 pmol/mg of protein, respectively. The Hill coefficient was 1.0, indicating a single entity of binding site and no cooperativity. The binding site was highly specific for iloprost among PGs tested (iloprost much greater than PGE1 greater than carbacyclin greater than PGE2). In contrast, the membrane fraction had the binding site specific for PGE2 and PGE1, which was distinct from the prostacyclin receptor. The dissociation of bound (3H)iloprost from the membrane fraction was specifically enhanced by guanine nucleotides. Furthermore, iloprost dose-dependently enhanced the activity of adenylate cyclase in a GTP-dependent manner. These results indicate that a specific prostacyclin receptor is coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mastocytoma cells.

  16. High-resolution definition of vaccine-elicited B cell responses against the HIV primary receptor binding site.

    Science.gov (United States)

    Sundling, Christopher; Li, Yuxing; Huynh, Nick; Poulsen, Christian; Wilson, Richard; O'Dell, Sijy; Feng, Yu; Mascola, John R; Wyatt, Richard T; Karlsson Hedestam, Gunilla B

    2012-07-11

    The high overall genetic homology between humans and rhesus macaques, coupled with the phenotypic conservation of lymphocyte populations, highlights the potential use of nonhuman primates (NHPs) for the preclinical evaluation of vaccine candidates. For HIV-1, experimental models are needed to identify vaccine regimens capable of eliciting desired immune responses, such as broadly neutralizing antibodies (bNAbs). One important neutralization target on the HIV-1 envelope glycoproteins (Envs) is the conserved primary CD4 receptor binding site (CD4bs). The isolation and characterization of CD4bs-specific neutralizing monoclonal Abs (mAbs) from HIV-1-infected individuals have provided insights into how broadly reactive Abs target this conserved epitope. In contrast, and for reasons that are not understood, current Env immunogens elicit CD4bs-directed Abs with limited neutralization breadth. To facilitate the use of the NHP model to address this and other questions relevant to human humoral immunity, we defined features of the rhesus macaque immunoglobulin (Ig) loci and compared these to the human Ig loci. We then studied Env-immunized rhesus macaques, identified single B cells expressing CD4bs-specific Abs, and sequenced and expressed a panel of functional mAbs. Comparison of vaccine-elicited mAbs with HIV-1 infection-induced mAbs revealed differences in the degree of somatic hypermutation of the Abs as well as in the fine specificities targeted within the CD4bs. These data support the use of the preclinical NHP model to characterize vaccine-induced B cell responses at high resolution.

  17. Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells.

    Science.gov (United States)

    Chen, F; Figueroa, D J; Marmorstein, A D; Zhang, Q; Petrukhin, K; Caskey, C T; Austin, C P

    1999-12-21

    In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814-4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription-PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of approximately 7.5 kb, approximately 3.0 kb, and approximately 2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.

  18. Study of binding glycyrrhetic acid to AT1 receptor

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  19. A computational analysis of non-genomic plasma membrane progestin binding proteins: signaling through ion channel-linked cell surface receptors.

    Science.gov (United States)

    Morrill, Gene A; Kostellow, Adele B; Gupta, Raj K

    2013-12-11

    A number of plasma membrane progestin receptors linked to non-genomic events have been identified. These include: (1) α1-subunit of the Na(+)/K(+)-ATPase (ATP1A1), (2) progestin binding PAQR proteins, (3) membrane progestin receptor alpha (mPRα), (4) progesterone receptor MAPR proteins and (5) the association of nuclear receptor (PRB) with the plasma membrane. This study compares: the pore-lining regions (ion channels), transmembrane (TM) helices, caveolin binding (CB) motifs and leucine-rich repeats (LRRs) of putative progesterone receptors. ATP1A1 contains 10 TM helices (TM-2, 4, 5, 6 and 8 are pores) and 4 CB motifs; whereas PAQR5, PAQR6, PAQR7, PAQRB8 and fish mPRα each contain 8 TM helices (TM-3 is a pore) and 2-4 CB motifs. MAPR proteins contain a single TM helix but lack pore-lining regions and CB motifs. PRB contains one or more TM helices in the steroid binding region, one of which is a pore. ATP1A1, PAQR5/7/8, mPRα, and MAPR-1 contain highly conserved leucine-rich repeats (LRR, common to plant membrane proteins) that are ligand binding sites for ouabain-like steroids associated with LRR kinases. LRR domains are within or overlap TM helices predicted to be ion channels (pore-lining regions), with the variable LRR sequence either at the C-terminus (PAQR and MAPR-1) or within an external loop (ATP1A1). Since ouabain-like steroids are produced by animal cells, our findings suggest that ATP1A1, PAQR5/7/8 and mPRα represent ion channel-linked receptors that respond physiologically to ouabain-like steroids (not progestin) similar to those known to regulate developmental and defense-related processes in plants.

  20. Does a nonclassical signaling mechanism underlie an increase of estradiol-mediated gonadotropin-releasing hormone receptor binding in ovine pituitary cells?

    Science.gov (United States)

    Davis, Tracy L; Whitesell, Jennifer D; Cantlon, Jeremy D; Clay, Colin M; Nett, Terry M

    2011-10-01

    Estradiol-17beta (E2) is the major regulator of GnRH receptor (GnRHR) gene expression and number during the periovulatory period; however, the mechanisms underlying E2 regulation of the GNRHR gene remain undefined. Herein, we find that E2 conjugated to BSA (E2-BSA) mimics the stimulatory effect of E2 on GnRH binding in primary cultures of ovine pituitary cells. The time course for maximal GnRH analog binding was similar for both E2 and E2-BSA. The ability of E2 and E2-BSA to increase GnRH analog binding was blocked by the estrogen receptor (ER) antagonist ICI 182,780. Also, increased GnRH analog binding in response to E2 and the selective ESR1 agonist propylpyrazole triol was blocked by expression of a dominant-negative form of ESR1 (L540Q). Thus, membrane-associated ESR1 is the likely candidate for mediating E2 activation of the GNRHR gene. As cAMP response element binding protein (CREB) is an established target for E2 activation in gonadotrophs, we next explored a potential role for this protein as an intracellular mediator of the E2 signal. Consistent with this possibility, adenoviral-mediated expression of a dominant-negative form of CREB (A-CREB) completely abolished the ability of E2 to increase GnRH analog binding in primary cultures of ovine pituitary cells. Finally, the presence of membrane-associated E2 binding sites on ovine pituitary cells was demonstrated using a fluorescein isothiocyanate conjugate of E2-BSA. We suggest that E2 regulation of GnRHR number during the preovulatory period reflects a membrane site of action and may proceed through a nonclassical signaling mechanism, specifically a CREB-dependent pathway.

  1. Phytoestrogens genistein and daidzin enhance the acetylcholinesterase activity of the rat pheochromocytoma cell line PC12 by binding to the estrogen receptor.

    Science.gov (United States)

    Isoda, Hiroko; Talorete, Terence P N; Kimura, Momoko; Maekawa, Takaaki; Inamori, Yuhei; Nakajima, Nobuyoshi; Seki, Humitake

    2002-11-01

    Some compounds derived from plants have been known to possess estrogenic properties and can thus alter the physiology of higher organisms. Genistein and daidzin are examples of these phytoestrogens, which have recently been the subject of extensive research. In this study, genistein and daidzin were found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at concentrations as low as 0.08 muM by binding to the estrogen receptor (ER). Results have shown that this enhancement was effectively blocked by the known estrogen receptor antagonist tamoxifen, indicating the involvement of the ER in AChE induction. That genistein and daidzin are estrogenic were confirmed in a cell proliferation assay using the human breast cancer cell line MCF7. This proliferation was also blocked by tamoxifen, again indicating the involvement of the ER. On the other hand, incubating the PC12 cells in increasing concentrations of 17 beta-estradiol (E2) did not lead to enhanced AChE activity, even in the presence of genistein or daidzin. This suggests that mere binding of an estrogenic compound to the ER does not necessarily lead to enhanced AChE activity. Moreover, the effect of the phytoestrogens on AChE activity cannot be expressed in the presence of E2 since they either could not compete with the natural ligand in binding to the ER or that E2 down-regulates its own receptor. This study clearly suggests that genistein and daidzin enhance AChE activityin PC12 cells by binding to the ER; however, the actual mechanism of enhancement is not known.

  2. Polymorphisms of the T cell receptor CD3delta and CD3varepsilon chains affect anti-CD3 antibody binding and T cell activation

    DEFF Research Database (Denmark)

    Boding, Lasse; Nielsen, Martin Weiss; Bonefeld, Charlotte Menné

    2010-01-01

    T cell receptor (TCR) structure and function have been thoroughly studied for decades. Production and analyses of knock-out and knock-in mice with mutations in the CD3 chains have contributed significantly to these studies. The generation of such gene-modified mice relies on the availability...... result in amino acid substitutions in the ectodomains of both the CD3delta and CD3varepsilon chains in 129 mice compared to C57BL/6 mice. The amino acid substitutions do not change the stoichiometry or surface expression level of the TCR complex in 129 T cells but cause reduced anti-CD3 antibody binding...... to 129 T cells. Further, when stimulated with mitogenic anti-CD3 antibodies, T cells from the 129 strains show reduced expression of the activation marker CD69, Ca(2+) flux, IL-2 production and proliferative responses compared to C57BL/6 T cells. These findings demonstrate that polymorphisms of the CD3...

  3. An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis.

    Science.gov (United States)

    Samten, Buka; Townsend, James C; Sever-Chroneos, Zvjezdana; Pasquinelli, Virginia; Barnes, Peter F; Chroneos, Zissis C

    2008-07-01

    Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.

  4. Crosstalk between the peroxisome proliferator-activated receptor γ (PPARγ) and the vitamin D receptor (VDR) in human breast cancer cells: PPARγ binds to VDR and inhibits 1α,25-dihydroxyvitamin D3 mediated transactivation.

    Science.gov (United States)

    Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang; Mehta, Rajeshwari R; von Knethen, Andreas; Choubey, Divaker; Mehta, Rajendra G

    2012-11-15

    Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ERα) physically binds to peroxisome proliferator-activated receptor gamma (PPARγ) and inhibits its transcriptional activity. The interaction between PPARγ and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPARγ and VDR signaling, and for the first time we show that PPARγ physically associates with VDR in human breast cancer cells. We found that overexpression of PPARγ decreased 1α,25-dihydroxyvitamin D(3) (1,25D(3)) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPARγ's hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPARγ's AF2 domain attenuated its repressive action on 1,25D(3) transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPARγ was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXRα). Overexpression of RXRα blocked PPARγ's suppressive effect on 1,25D(3) action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPARγ and VDR pathways.

  5. Receptor binding profile of Otilonium bromide.

    Science.gov (United States)

    Evangelista, S; Giachetti, A; Chapelain, B; Neliat, G; Maggi, C A

    1998-08-01

    The interaction of Otilonium bromide (OB) with binding sites for 63 different receptors and ion channels in appropriate preparations has been investigated. Experiments were also performed in rat colon, the preferred tissue for OB 'in vivo' uptake after oral administration. Among the receptors investigated OB binds with sub microM affinity to muscarinic M1, M2, M4, M5 and PAF receptors and with microM affinity to the diltiazem binding site on L type Ca2+ channels. In the rat colon OB shows competitive interaction with the verapamil binding site on L type Ca2+ channels and with muscarinic M2 receptors with IC50 of 1020 and 1220 nM, respectively. These findings provide a molecular rationale to explain the spasmolytic action exerted by OB on intestinal smooth muscle. In particular, a combination of antimuscarinic and Ca2+ channel blocker properties seems to best account for the action of this compound.

  6. A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor gene expression

    Science.gov (United States)

    Falvo, James V.; Brinkman, Brigitta M. N.; Tsytsykova, Alla V.; Tsai, Eunice Y.; Yao, Tso-Pang; Kung, Andrew L.; Goldfeld, Anne E.

    2000-04-01

    The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor (TNF-) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF- gene induction by TCR activation is inhibited, whereas virus induction of the TNF- gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-α gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

  7. HLA-Cw*0102-restricted HIV-1 p24 epitope variants can modulate the binding of the inhibitory KIR2DL2 receptor and primary NK cell function.

    Directory of Open Access Journals (Sweden)

    Lena Fadda

    Full Text Available Accumulating evidence suggests an important role for Natural Killer (NK cells in the control of HIV-1 infection. Recently, it was shown that NK cell-mediated immune pressure can result in the selection of HIV-1 escape mutations. A potential mechanism for this NK cell escape is the selection of HLA class I-presented HIV-1 epitopes that allow for the engagement of inhibitory killer cell immunoglobulin-like receptors (KIRs, notably KIR2DL2. We therefore investigated the consequences of sequence variations within HLA-Cw*0102-restricted epitopes on the interaction of HLA-Cw*0102 with KIR2DL2 using a large panel of overlapping HIV-1 p24 Gag peptides. 217 decameric peptides spanning the HIV-1 p24 Gag consensus sequence were screened for HLA-Cw*0102 stabilization by co-incubation with Cw*0102⁺/TAP-deficient T2 cells using a flow cytometry-based assay. KIR2DL2 binding was assessed using a KIR2DL2-IgG fusion construct. Function of KIR2DL2⁺ NK cells was flow cytometrically analyzed by measuring degranulation of primary NK cells after co-incubation with peptide-pulsed T2 cells. We identified 11 peptides stabilizing HLA-Cw*0102 on the surface of T2 cells. However, only one peptide (p24 Gag₂₀₉₋₂₁₈ AAEWDRLHPV allowed for binding of KIR2DL2. Notably, functional analysis showed a significant inhibition of KIR2DL2⁺ NK cells in the presence of p24 Gag₂₀₉₋₂₁₈-pulsed T2 cells, while degranulation of KIR2DL2⁻ NK cells was not affected. Moreover, we demonstrated that sequence variations in position 7 of this epitope observed frequently in naturally occurring HIV-1 sequences can modulate binding to KIR2DL2. Our results show that the majority of HIV-1 p24 Gag peptides stabilizing HLA-Cw*0102 do not allow for binding of KIR2DL2, but identified one HLA-Cw*0102-presented peptide (p24 Gag₂₀₉₋₂₁₈ that was recognized by the inhibitory NK cell receptor KIR2DL2 leading to functional inhibition of KIR2DL2-expressing NK cells

  8. Inhibition of cell-cell binding by lipid assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, Jon O. (Rodeo, CA); Bargatze, Robert F. (Bozeman, MT)

    2001-05-22

    This invention relates generally to the field of therapeutic compounds designed to interfere between the binding of ligands and their receptors on cell surface. More specifically, it provides products and methods for inhibiting cell migration and activation using lipid assemblies with surface recognition elements that are specific for the receptors involved in cell migration and activation.

  9. CART (cocaine- and amphetamine-regulated transcript) peptide specific binding sites in PC12 cells have characteristics of CART peptide receptors.

    Science.gov (United States)

    Nagelová, Veronika; Pirník, Zdeno; Železná, Blanka; Maletínská, Lenka

    2014-02-14

    CART (cocaine- and amphetamine-regulated transcript) peptide is a neuropeptide with a powerful central anorexigenic effect. Specific CART peptide binding sites, most likely CART peptide receptors, have been found in PC12 cells. This study further characterizes the CART peptide binding sites in PC12 cells. After differentiation to a neuronal phenotype with nerve growth factor, the number of CART peptide binding sites in PC12 cells tripled. Following dexamethasone treatment, which transforms PC12 cells into chromaffin-like cells, the number of CART peptide binding sites substantially decreased. CART peptide did not affect the differentiation or acetylcholinesterase activity of PC12 cells, indicating that CART peptide does not participate in differentiation or neuronal activity. CART peptide increased the phosphorylation of SAPK/JNK (stress-activated protein kinase/c-Jun-amino-terminal kinase) and subsequent c-Jun protein expression. These effects were reversed by SP600125, a specific JNK-kinase inhibitor. CART peptide did not significantly affect ERK (extracellular signal-regulated kinase), CREB (cAMP responsive element binding protein), or p38 phosphorylation and c-Fos protein expression. Central administration of CART peptide into mice also resulted in increased c-Jun positive cells in dorsomedial hypothalamic nucleus and nucleus of the solitary tract, areas involved in food intake regulation. Activation of c-Jun by CART peptide might indicate a possible role of CART peptide in managing stress conditions rather than a role in cell proliferation or differentiation as well as the more complex and/or specific regulation ways by transcription factors in some nuclei involved in food intake regulation. The characteristics of stress that CART peptide potentially mediates should be further studied.

  10. The impact of the 67kDa laminin receptor on both cell-surface binding and anti-allergic action of tea catechins.

    Science.gov (United States)

    Fujimura, Yoshinori; Umeda, Daisuke; Yamada, Koji; Tachibana, Hirofumi

    2008-08-15

    Here, we investigated the structure-activity relationship of major green tea catechins and their corresponding epimers on cell-surface binding and inhibitory effect on histamine release. Galloylated catechins; (-)-epigallocatechin-3-O-gallate (EGCG), (-)-gallocatechin-3-O-gallate (GCG), (-)-epicatechin-3-O-gallate (ECG), and (-)-catechin-3-O-gallate (CG) showed the cell-surface binding to the human basophilic KU812 cells by surface plasmon resonance analysis, but their non-galloylated forms did not. Binding activities of pyrogallol-type catechins (EGCG and GCG) were higher than those of catechol-type catechins (ECG and CG). These patterns were also observed in their inhibitory effects on histamine release. Previously, we have reported that biological activities of EGCG are mediated through the binding to the cell-surface 67kDa laminin receptor (67LR). Downregulation of 67LR expression caused a reduction of both activities of galloylated catechins. These results suggest that both the galloyl moiety and the B-ring hydroxylation pattern contribute to the exertion of biological activities of tea catechins and their 67LR-dependencies.

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

    Science.gov (United States)

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

    2014-04-17

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

  12. ABP: a novel AMPA receptor binding protein.

    Science.gov (United States)

    Srivastava, S; Ziff, E B

    1999-04-30

    We review the cloning of a novel AMPA receptor binding protein (ABP) that interacts with GluR2/3 and is homologous to GRIP. ABP is enriched in the PSD with GluR2 and is localized to the PSD by EM. ABP binds GluR2 via the C-terminal VXI motif through a Class I PDZ interaction. ABP and GRIP can also homo- and heteromultimerize. Thus, ABP and GRIP may be involved in AMPA receptor regulation and localization, by linking it to other cytoskeletal or signaling molecules. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors. We are currently investigating proteins that bind ABP and that may regulate the AMPA receptor.

  13. Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

    Science.gov (United States)

    Godoi, Paulo H C; Wilkie-Grantham, Rachel P; Hishiki, Asami; Sano, Renata; Matsuzawa, Yasuko; Yanagi, Hiroko; Munte, Claudia E; Chen, Ya; Yao, Yong; Marassi, Francesca M; Kalbitzer, Hans R; Matsuzawa, Shu-Ichi; Reed, John C

    2016-07-01

    B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells.

    Science.gov (United States)

    Shafren, D R; Williams, D T; Barry, R D

    1997-12-01

    The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.

  15. Phytoestrogens genistein and daidzin enhance the acetylcholinesterase activity of the rat pheochromocytoma cell line PC12 by binding to the estrogen receptor

    OpenAIRE

    Isoda, Hiroko; Talorete, Terence P. N.; Kimura, Momoko; Maekawa, Takaaki; Inamori, Yuhei; Nakajima, Nobuyoshi; Seki, Humitake

    2002-01-01

    Some compounds derived from plants have been known to possess estrogenic properties and can thus alter the physiology of higher organisms. Genistein and daidzin are examples of these phytoestrogens, which have recently been the subject of extensive research. In this study, genistein and daidzin were found to enhance the acetylcholinesterase (AChE) activity of the rat neuronal cell line PC12 at concentrations as low as 0.08 μM by binding to the estrogen receptor (ER). Results have shown that t...

  16. Leptospiral outer membrane lipoprotein LipL32 binding on toll-like receptor 2 of renal cells as determined with an atomic force microscope.

    Science.gov (United States)

    Hsu, Shen-Hsing; Lo, Yueh-Yu; Tung, Jung-Yu; Ko, Yi-Ching; Sun, Yuh-Ju; Hung, Cheng-Chieh; Yang, Chih-Wei; Tseng, Fan-Gang; Fu, Chien-Chung; Pan, Rong-Long

    2010-07-06

    Leptopirosis is a renal disease caused by pathogenic Leptospira that primarily infects the renal proximal tubules, consequently resulting in severe tubular injuries and malfunctions. The protein extracted from the outer membrane of this pathogenic strain contains a major component of a 32 kDa lipoprotein (LipL32), which is absent in the counter membrane of nonpathogenic strains and has been identified as a crucial factor for host cell infection. Previous studies showed that LipL32 induced inflammatory responses and interacted with the extracellular matrix (ECM) of the host cell. However, the exact relationship between LipL32-mediated inflammatory responses and ECM binding is still unknown. In this study, an atomic force microscope with its tip modified by purified LipL32 was used to assess the interaction between LipL32 and cell surface receptors. Furthermore, an antibody neutralization technique was employed to identify Toll-like receptor 2 (TLR2) but not TLR4 as the major target of LipL32 attack. The interaction force between LipL32 and TLR2 was measured as approximately 59.5 +/- 8.7 pN, concurring with the theoretical value for a single-pair molecular interaction. Moreover, transformation of a TLR deficient cell line with human TLR2 brought the interaction force from the basal level to approximately 60.4 +/- 11.5 pN, confirming unambiguously TLR2 as counter receptor for LipL32. The stimulation of CXCL8/IL-8 expression by full-length LipL32 as compared to that without the N-terminal signal peptide domain suggests a significant role of the signal peptide of the protein in the inflammatory responses. This study provides direct evidence that LipL32 binds to TLR2, but not TLR4, on the cell surface, and a possible mechanism for the virulence of leptospirosis is accordingly proposed.

  17. DC-SIGN:binding receptors for hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    王全楚; 冯志华; 聂青和; 周永兴

    2004-01-01

    Objective To review the recent developments in and research into binding receptors of hepatitis C virus (HCV) and especially the role of dendritic cell-specitic adhesion receptor (DC-SIGN) in HCV.Data sources Both Chinese- and English-languge literature was searched using MEDLINE (2000-2003) and the databank of Chinese-language literature (2000-2003).Study selection Relevant articles on DC-SIGN and HCV binding receptors in recent domestic and foreign literature were selected.Data extraction Data were mainly extracted from 40 articles which are listed in the references section of this review. Results DC-SIGN, a dendritic cell-specific adhesion receptor and a type Ⅱ transmembrane mannose-binding C-type lectin, is very important in the function of dendritic cells (DC), both in mediating na(I)ve T cell interactions through ICAM-3 and as a rolling receptor that mediates the DC-specific ICAM-2-dependent migration processes-It can be used by HCV and other viral and bacterial pathogens including human immunodeficiency virus (HIV), Ebola virus, CMV and Mycobacterium tuberculosis- to facilitate infection. Both DC-SIGN and DC-SIGNR can act either in cis, by concentrating virus on target cells, or in trans, by transmission of bound virus to a target cell expressing appropriate entry receptors. Recent report showed that DC-S IGN not only plays a role in entry into DC, HCV E2 interaction with DC-SIGN might also be detrimental to the interaction of DC with T cells during antigen presentation.Conclusions DC-SIGNs are high-affinity binding receptors for HCV.The clinical strategies that target DC-SIGN may be successful in restricting HCV dissemination and pathogenesis as well as directing the migration of DCs to manipulate appropriate immune responses in autoimmunity and tumorigenic situations.

  18. Enhanced human receptor binding by H5 haemagglutinins

    OpenAIRE

    Xiong, Xiaoli; Xiao, Haixia; Martin, Stephen R.; Coombs, Peter J.; Liu, Junfeng; Collins, Patrick J.; Vachieri, Sebastien G.; Walker, Philip A.; Lin, Yi Pu; McCauley, John W.; Gamblin, Steven J.; John J Skehel

    2014-01-01

    Mutant H5N1 influenza viruses have been isolated from humans that have increased human receptor avidity. We have compared the receptor binding properties of these mutants with those of wild-type viruses, and determined the structures of their haemagglutinins in complex with receptor analogues. Mutants from Vietnam bind tighter to human receptor by acquiring basic residues near the receptor binding site. They bind more weakly to avian receptor because they lack specific interactions between As...

  19. [Extracellular Y-box binding protein-1 promotes proliferation and metastasis of HepG2 cells through Notch3 receptor].

    Science.gov (United States)

    Shi, J; Li, P; Zou, L; Chen, P; Zhang, L P

    2016-03-20

    To clarify whether HepG2 cells actively secrete Y-box binding protein-1 (YB-1) under stress conditions, and to investigate the pathological significance and mechanism of action of extracellular YB-1. HepG2 cells were stimulated and treated by gradient concentrations of lipopolysaccharide (LPS) and adriamycin, the supernatant of the culture solution was collected by centrifugation, and the established chemiluminescence immunoassay (CLIA) was used for real-time quantitative determination of YB-1 level in the supernatant. The co-immunoprecipitation assay was used to detect whether extracellular YB-1 specifically bound to Notch3 receptor, and Western blot was used to measure the expression of Notch-NICD. The gradient concentrations of recombinant YB-1 were co-cultured with HepG2 cells, and MTT and migration assays were used to analyze the proliferation and invasion/metastasis of HepG2 cells. One-way analysis of variance was used for comparison of data between multiple groups. The results of CLIA confirmed that the level of extracellular YB-1 in the supernatant was significantly higher than that in the control group (F= 10.54,PHepG2 cells (F= 9.405,PHepG2 cells can actively secrete YB-1 via non-classical pathways. Extracellular YB-1 can specifically bind to Notch3 receptor and further up-regulate its expression, and then promote the proliferation and invasion/metastasis of HepG2 cells. This study lays a foundation for further clarifying the pathogenesis of hepatocellular carcinoma and investigating the biological relationship between extracellular YB-1 and malignant tumors.

  20. TNF receptor-associated factor-2 binding site is involved in TNFR75-dependent enhancement of TNFR55-induced cell death

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    TNF recepter-55 is the main mediator of TNF-induced apoptosis. TNF receptor-75-dependent induction or enhancement of cytotoxicity has been explained by intracellular signaling, “ligand passing”, or induction of endogenous TNF. To study the function of human TNF receptor-75 (hTR75) and the interaction between human TNF receptor-55 (hTR55) and hTR75 in hTNFc-induced cytotoxicity, Hep-2 cells were transfected with bicistronic expression vector of hTR75 and its deletion mutants genes. hTNFα-induced cytotoxicity was determined by crystal violet colorimetric method. The expression of hTR75 and its deletion mutants in Hep-2 cells was demonstrated by RT-PCR and indirect ELISA. We found that the overexpressed hTR75 could significantly increase the susceptibility of Hep-2 cells to hTNFα which especiαlly required TRAF2 binding site. hTR75 could not only mediate partial hTNFα-induced cytotoxicity independently but also fulfill an accessory role in enhancing or synergizing hTR55-mediated cytotoxicity.

  1. Inhibitory effect of acetyl-11-keto-beta-boswellic acid on androgen receptor by interference of Sp1 binding activity in prostate cancer cells.

    Science.gov (United States)

    Yuan, Hui-Qing; Kong, Feng; Wang, Xiao-Ling; Young, Charles Y F; Hu, Xiao-Yan; Lou, Hong-Xiang

    2008-06-01

    Androgen receptor (AR)-mediated signaling is crucial for the development and progression of prostate cancer (PCa). Naturally occurring phytochemicals that target the AR signaling offer significant protection against this disease. Acetyl-11-keto-beta-boswellic acid (AKBA), a compound isolated from the gum-resin of Boswellia carterii, caused G1-phase cell cycle arrest with an induction of p21(WAF1/CIP1), and a reduction of cyclin D1 as well in prostate cancer cells. AKBA-mediated cellular proliferation inhibition was associated with a decrease of AR expression at mRNA and protein levels. Furthermore, the functional biomarkers used in evaluation of AR transactivity showed suppressions of prostate-specific antigen promoter-dependent and androgen responsive element-dependent luciferase activities. Additionally, down-regulation of an AR short promoter mainly containing a Sp1 binding site suggested the essential role of Sp1 for the reduction of AR expression in cells exposed to AKBA. Interruption effect of AKBA on Sp1 binding activity but not Sp1 protein levels was further confirmed by EMSA and transient transfection with a luciferase reporter driven by three copies of the Sp1 binding site of the AR promoter. Therefore, anti-AR properties ascribed to AKBA suggested that AKBA-containing drugs could be used for the development of novel therapeutic chemicals.

  2. Development of prolactin receptor antagonists with reduced pH-dependence of receptor binding

    DEFF Research Database (Denmark)

    Hansen, Mathilde Johanne Kaas; Olsen, Johan Gotthardt; Bernichtein, Sophie;

    2011-01-01

    H than at physiological pH and since the extracellular environment around solid tumors often is acidic, it is desirable to develop antagonists that have improved binding affinity at low pH. The pK(a) value of a histidine side chain is ~6.8 making histidine residues obvious candidates for examination....... From evaluation of known molecular structures of human prolactin, of the prolactin receptor and of different complexes of the two, three histidine residues in the hormone-receptor binding site 1 were selected for mutational studies. We analyzed 10 variants by circular dichroism spectroscopy, affinity...... and thermodynamic characterization of receptor binding by isothermal titration calorimetry combined with in vitro bioactivity in living cells. Histidine residue 27 was recognized as a central hot spot for pH sensitivity and conservative substitutions at this site resulted in strong receptor binding at low pH. Pure...

  3. Identification of a specific region of Plasmodium falciparum EBL-1 that binds to host receptor glycophorin B and inhibits merozoite invasion in human red blood cells.

    Science.gov (United States)

    Li, Xuerong; Marinkovic, Marina; Russo, Crystal; McKnight, C James; Coetzer, Theresa L; Chishti, Athar H

    2012-05-01

    The malaria parasite Plasmodium falciparum invades human erythrocytes through multiple pathways utilizing several ligand-receptor interactions. These interactions are broadly classified in two groups according to their dependency on sialic acid residues. Here, we focus on the sialic acid-dependent pathway by using purified glycophorins and red blood cells (RBCs) to screen a cDNA phage display library derived from P. falciparum FCR3 strain, a sialic acid-dependent strain. This screen identified several parasite proteins including the erythrocyte-binding ligand-1, EBL-1. The phage cDNA insert encoded the 69-amino acid peptide, termed F2i, which is located within the F2 region of the DBL domain, designated here as D2, of EBL-1. Recombinant D2 and F2i polypeptides bound to purified glycophorins and RBCs, and the F2i peptide was found to interfere with binding of D2 domain to its receptor. Both D2 and F2i polypeptides bound to trypsin-treated but not neuraminidase or chymotrypsin-treated erythrocytes, consistent with known glycophorin B resistance to trypsin, and neither the D2 nor F2i polypeptide bound to glycophorin B-deficient erythrocytes. Importantly, purified D2 and F2i polypeptides partially inhibited merozoite reinvasion in human erythrocytes. Our results show that the host erythrocyte receptor glycophorin B directly interacts with the DBL domain of parasite EBL-1, and the core binding site is contained within the 69 amino acid F2i region (residues 601-669) of the DBL domain. Together, these findings suggest that a recombinant F2i peptide with stabilized structure could provide a protective function at blood stage infection and represents a valuable addition to a multi-subunit vaccine against malaria. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

    Energy Technology Data Exchange (ETDEWEB)

    Kikuchi, M.; Ishii, S. (Waseda Univ., Tokyo (Japan))

    1989-12-01

    By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of (1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; (2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and (3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final {sup 125}I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. {sup 125}I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

  5. A Highly Tilted Binding Mode by a Self-Reactive T Cell Receptor Results in Altered Engagement of Peptide and MHC

    Energy Technology Data Exchange (ETDEWEB)

    D Sethi; D Schubert; A Anders; A Heroux; D Bonsor; C Thomas; E Sundberg; J Pyrdol; K Wucherpfennig

    2011-12-31

    Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-{alpha} chain with the MHC class II {beta} chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3{beta} loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3{alpha} loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

  6. A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC

    Energy Technology Data Exchange (ETDEWEB)

    Sethi, D.K.; Heroux, A.; Schubert, D. A.; Anders, A.-K.; Bonsor, D. A.; Thomas, C. P.; Sundberg, E. J.; Pyrdol, J.; Wucherpfennig, K. W.

    2011-01-17

    Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-{alpha} chain with the MHC class II {beta} chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3{beta} loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3{alpha} loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

  7. Requirement of plasminogen binding to its cell-surface receptor α-enolase for efficient regeneration of normal and dystrophic skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Àngels Díaz-Ramos

    Full Text Available Adult regenerative myogenesis is central for restoring normal tissue structure and function after muscle damage. In muscle repair after injury, as in severe myopathies, damaged and necrotic fibers are removed by infiltrating inflammatory cells and then replaced by muscle stem cells or satellite cells, which will fuse to form new myofibers. Extracellular proteolysis mediated by uPA-generated plasmin plays a critical role in controlling inflammation and satellite-cell-dependent myogenesis. α-enolase has been described as plasminogen receptor in several cell types, where it acts concentrating plasmin proteolytic activity on the cell surface. In this study, we investigated whether α-enolase plasminogen receptor plays a regulatory role during the muscular repair process. Inhibitors of α-enolase/plasminogen binding: MAb11G1 (a monoclonal antibody against α-enolase and ε-aminocaproic acid, EACA (a lysine analogue inhibited the myogenic abilities of satellite cells-derived myoblasts. Furthermore, knockdown of α-enolase decreased myogenic fusion of myoblasts. Injured wild-type mice and dystrophic mdx mice were also treated with MAb11G1 and EACA. These treatments had negative impacts on muscle repair impairing satellite cell functions in vitro in agreement with blunted growth of new myofibers in vivo. Furthermore, both MAb11G1 and EACA treatments impaired adequate inflammatory cell infiltration and promoted extracellular matrix deposition in vivo, which resulted in persistent degeneration. These results demonstrate the novel requirement of α-enolase for restoring homeostasis of injured muscle tissue, by controlling the pericellular localization of plasmin activity.

  8. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies.

    Science.gov (United States)

    McGuire, Andrew T; Hoot, Sam; Dreyer, Anita M; Lippy, Adriana; Stuart, Andrew; Cohen, Kristen W; Jardine, Joseph; Menis, Sergey; Scheid, Johannes F; West, Anthony P; Schief, William R; Stamatatos, Leonidas

    2013-04-08

    Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti-CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.

  9. Chicken NK cell receptors.

    Science.gov (United States)

    Straub, Christian; Neulen, Marie-Luise; Sperling, Beatrice; Windau, Katharina; Zechmann, Maria; Jansen, Christine A; Viertlboeck, Birgit C; Göbel, Thomas W

    2013-11-01

    Natural killer cells are innate immune cells that destroy virally infected or transformed cells. They recognize these altered cells by a plethora of diverse receptors and thereby differ from other lymphocytes that use clonally distributed antigen receptors. To date, several receptor families that play a role in either activating or inhibiting NK cells have been identified in mammals. In the chicken, NK cells have been functionally and morphologically defined, however, a conclusive analysis of receptors involved in NK cell mediated functions has not been available. This is partly due to the low frequencies of NK cells in blood or spleen that has hampered their intensive characterization. Here we will review recent progress regarding the diverse NK cell receptor families, with special emphasis on novel families identified in the chicken genome with potential as chicken NK cell receptors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Receptor discrimination and control of agonist-antagonist binding.

    Science.gov (United States)

    Tallarida, R J

    1995-08-01

    The law of mass action is the common model for the interaction of agonist and antagonist compounds with cellular receptors. Parameters of the interaction, obtained from functional and radioligand-binding studies, allow discrimination and subtyping of receptors and aid in understanding specific mechanisms. This article reviews the theory and associated mathematical models and graphical transformations of data that underlie the determination of receptor parameters. The main theory assumes that agonist and antagonist compounds bind to cells that have a fixed number of receptors and provides the framework for obtaining drug-receptor parameters from data and their graphical transformations. Conditions that produce a change in receptor number, a newer concept in pharmacology, can have an important effect on the parameter values derived in the usual way. This review concludes with a discussion of the quantitative study of receptor-mediated feedback control of endogenous ligands, a very new topic with potentially important implications for understanding antagonist effectiveness, loss of control, and chaos in regulated mass action binding.

  11. Glucocorticoid receptors in murine erythroleukaemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Hammond, K.D.; Torrance, J.M.; DiDomenico, M.

    1987-01-01

    Glucocorticoid receptors in murine erythroleukaemic cells were studied in relation to hexamethylene bisacetamide (HMBA) induced differentiation. Specific binding of dexamethasone was measured. A single class of saturable, high affinity binding sites was demonstrated in intact cells; with cell homogenates or fractions binding was low and could not be reliably quantified. Receptor binding in whole cell suspensions was lower in cells which had been treated with HMBA (36.5 +/- 8.2 pmol/g protein) than in untreated controls (87.9 +/- 23.6 pmol/g protein); dissociation constants were similar in treated (2.7 nM) and untreated cells (2.5 nM). Dexamethasone, hydrocortisone, corticosterone and progesterone competed with tritium-labelled dexamethasone for receptor binding sites; cortisone, deoxycorticosterone and oestradiol had little effect.

  12. Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin

    DEFF Research Database (Denmark)

    Wewer, U M; Liotta, L A; Jaye, M

    1986-01-01

    of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were...

  13. Growth hormone binding to specific receptors stimulates growth and function of cloned insulin-producing rat insulinoma RIN-5AH cells

    DEFF Research Database (Denmark)

    Billestrup, Nils; Martin, J M

    1985-01-01

    Binding of 125I-labeled human GH (hGH) to a cloned rat insulin-producing cell line RIN-5AH in monolayer culture was studied along with some physiological effects of the hormone on these cells. Binding was time and temperature dependent, and steady state binding was observed in 60 min at 37 C...

  14. Identification of Insulin-Like Growth Factor-I Receptor (IGF-IR Gene Promoter-Binding Proteins in Estrogen Receptor (ER-Positive and ER-Depleted Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Rive Sarfstein

    2010-03-01

    Full Text Available The insulin-like growth factor I receptor (IGF-IR has been implicated in the etiology of breast cancer. Overexpression of the IGF-IR gene is a typical feature of most primary breast cancers, whereas low IGF-IR levels are seen at advanced stages. Hence, evaluation of IGF-IR levels might be important for assessing prognosis. In the present study, we employed a proteomic approach based on DNA affinity chromatography followed either by mass spectroscopy (MS or Western blot analysis to identify transcription factors that may associate with the IGF-IR promoter in estrogen receptor (ER-positive and ER-depleted breast cancer cells. A biotinylated IGF-IR promoter fragment was bound to streptavidin magnetic beads and incubated with nuclear extracts of breast cancer cells. IGF-IR promoter-binding proteins were eluted with high salt and analyzed by MS and Western blots. Among the proteins that were found to bind to the IGF-IR promoter we identified zinc finger transcription factors Sp1 and KLF6, ER-, p53, c-jun, and poly (ADP-ribosylation polymerase. Furthermore, chromatin immune-precipitation (ChIP analysis confirmed the direct in vivo binding of some of these transcription factors to IGF-IR promoter DNA. The functional relevance of binding data was assessed by cotransfection experiments with specific expression vectors along with an IGF-IR promoter reporter. In summary, we identified nuclear proteins that are potentially responsible for the differential expression of the IGF-IR gene in ER-positive and ER-depleted breast cancer cells.

  15. 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.

  16. Novel experimental design for steady-state processes: a systematic Bayesian approach for enzymes, drug transport, receptor binding, continuous culture and cell transport kinetics.

    Science.gov (United States)

    Crabbe, M James C; Murphy, Emma F; Gilmour, Steven G

    2005-01-01

    We demonstrate that a Bayesian approach (the use of prior knowledge) to the design of steady-state experiments can produce major gains quantifiable in terms of information, productivity and accuracy of each experiment. Developing the use of Bayesian utility functions, we have used a systematic method to identify the optimum experimental designs for a number of kinetic model data sets. This has enabled the identification of trends between kinetic model types, sets of design rules and the key conclusion that such designs should be based on some prior knowledge of the kinetic model. We suggest an optimal and iterative method for selecting features of the design such as the substrate range, number of measurements and choice of intermediate points. The final design collects data suitable for accurate modelling and analysis and minimises the error in the parameters estimated. It is equally applicable to enzymes, drug transport, receptor binding, microbial culture and cell transport kinetics.

  17. A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation.

    Science.gov (United States)

    Maglinao, Maha; Eriksson, Magdalena; Schlegel, Mark K; Zimmermann, Stephanie; Johannssen, Timo; Götze, Sebastian; Seeberger, Peter H; Lepenies, Bernd

    2014-02-10

    Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties.

  18. GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

    Science.gov (United States)

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation.

  19. Chicken SLAMF4 (CD244, 2B4), a receptor expressed on thrombocytes, monocytes, NK cells, and subsets of αβ-, γδ- T cells and B cells binds to SLAMF2.

    Science.gov (United States)

    Straub, Christian; Neulen, Marie-Luise; Viertlboeck, Birgit C; Göbel, Thomas W

    2014-02-01

    The SLAM family of membrane receptors is involved in the regulation of immune responses by controlling cytokines production, cytotoxicity as well as cell development, differentiation and proliferation, but has only been described in chickens, recently. The aim of this study was to characterize the avian homologue to mammalian SLAMF4 (CD244, 2B4), a cell surface molecule which belongs to the SLAM family of membrane receptors. We generated a SLAMF4 specific monoclonal antibody (mab) designated 8C7 and analyzed the SLAMF4 expression on cells isolated from various lymphoid organs. Subsets of αβ and γδ T cells found in peripheral blood lymphocytes (PBL) and spleen coexpressed SLAMF4. The expression was restricted to CD8α(+) T cells, whereas CD4(+) T cells and all thymocytes showed little or no reactivity upon staining with the 8C7 mab. Blood and splenic γδ T cells could be further differentiated according to their expression levels of SLAMF4 into two and three subsets, respectively. SLAMF4 was absent from bursal and splenic B cells, however, it was expressed by a distinct fraction of circulating B cells that were characterized by high level expression of Bu1, Ig, and CD40. SLAMF4 was also present on NK cells isolated from intestine of adult chickens or embryonic splenocytes identified by their coexpression of the 28-4 NK cell marker. Moreover, SLAMF4 expression was found on thrombocytes and monocytes. The interaction of SLAMF4 with SLAMF2 was proven by a reporter assay and could be blocked with the 8C7 mab. In conclusion, the avian SLAMF4 expression markedly differs from mammals; it binds to SLAMF2 and will be an important tool to discriminate several γδ T cell subsets. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Increased, Durable B-Cell and ADCC Responses Associated with T-Helper Cell Responses to HIV-1 Envelope in Macaques Vaccinated with gp140 Occluded at the CD4 Receptor Binding Site.

    Science.gov (United States)

    Bogers, Willy M J M; Barnett, Susan W; Oostermeijer, Herman; Nieuwenhuis, Ivonne G; Beenhakker, Niels; Mortier, Daniella; Mooij, Petra; Koopman, Gerrit; Remarque, Edmund; Martin, Gregoire; Lai, Rachel Pei-Jen; Dey, Antu K; Sun, Yide; Burke, Brian; Ferrari, Guido; Montefiori, David; Martin, Loic; Davis, David; Srivastava, Indresh; Heeney, Jonathan L

    2017-10-01

    Strategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens (Ag) for more long-lived, efficacious HIV-1 vaccine-induced B-cell responses. HIV-1 Env gp140 (native or uncleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells results in anergic effects on T-cell recruitment and consequently a lack of strong, robust, and durable B-memory responses. To test this hypothesis, we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3-kDa CD4 miniprotein mimetic, serving to block ligation of gp140 on CD4(+) T cells while preserving CD4-inducible (CDi) neutralizing epitopes targeted by antibody-dependent cellular cytotoxicity (ADCC) effector responses. Importantly, immunization of rhesus macaques consistently gave superior B-cell (P T-helper enzyme-linked immunosorbent spot (ELISpot) assays of the CD4bs-occluded group increased earlier (P = 0.025) during the inductive phase. Importantly, CD4bs-occluded gp140 antigen induced superior B-cell and ADCC responses, and the elevated B-cell responses proved to be remarkably durable, lasting more than 60 weeks postimmunization.IMPORTANCE Attempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have been unsuccessful until now. Antigen-specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T cells as soluble shed antigen or as antigen-antibody complexes, causing impairment in the activation of these specialized CD4-positive T cells. We proposed that CD4-binding impairment is partly responsible for the relatively poor B-cell responses to HIV envelope-based vaccines. To test this hypothesis, we blocked the CD4 binding site of the envelope antigen and compared it to

  1. Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization

    Science.gov (United States)

    Barmina, O. Y.; Walling, H. W.; Fiacco, G. J.; Freije, J. M.; Lopez-Otin, C.; Jeffrey, J. J.; Partridge, N. C.

    1999-01-01

    We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

  2. The mitochondrial Na+/Ca2+exchanger may reduce high glucose-induced oxidative stress and nucleotide-binding oligomerization domain receptor 3 inflammasome activation in endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Yuan ZU; Li-Juan WAN; Shao-Yuan CUI; Yan-Ping GONG; Chun-Lin LI

    2015-01-01

    Background The mitochondrial Na+/Ca2+exchanger, NCLX, plays an important role in the balance between Ca2+influx and efflux across the mitochondrial inner membrane in endothelial cells. Mitochondrial metabolism is likely to be affected by the activity of NCLX because Ca2+activates several enzymes of the Krebs cycle. It is currently believed that mitochondria are not only centers of energy produc-tion but are also important sites of reactive oxygen species (ROS) generation and nucleotide-binding oligomerization domain receptor 3 (NLRP3) inflammasome activation. Methods&Results This study focused on NCLX function, in rat aortic endothelial cells (RAECs), induced by glucose. First, we detected an increase in NCLX expression in the endothelia of rats with diabetes mellitus, which was induced by an injection of streptozotocin. Next, colocalization of NCLX expression and mitochondria was detected using confocal analysis. Suppression of NCLX expression, using an siRNA construct (siNCLX), enhanced mitochondrial Ca2+influx and blocked efflux induced by glucose. Un-expectedly, silencing of NCLX expression induced increased ROS generation and NLRP3 inflammasome activation. Conclusions These findings suggest that NCLX affects glucose-dependent mitochondrial Ca2+signaling, thereby regulating ROS generation and NLRP3 in-flammasome activation in high glucose conditions. In the early stages of high glucose stimulation, NCLX expression increases to compensate in order to self-protect mitochondrial maintenance, stability, and function in endothelial cells.

  3. Inhibition Of Call-Cell Binding By Kipid Assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Nagy, Jon O. (Rodeo, CA), Bargatze, Robert F. (Bozeman, MT)

    2003-12-16

    This invention relates generally to the field of therapeutic compounds designed to interfere between the binding of ligands and their receptors on cell surface. More specifically, it provides products and methods for inhibiting cell migration and activation using lipid assemblies with surface recognition elements that are specific for the receptors involved in cell migration and activation.

  4. Receptor binding characteristics and cytotoxicity of insulin-methotrexate

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hong Ou; An-Ren Kuang; Zheng-Lu Liang; Xian Peng; Yu-Guo Zhong

    2004-01-01

    AIM: To characterize the receptor binding affinity and cytotoxicity of insulin-methotrexate (MTX) for the potential utilization of insulin as carriers for carcinoma target drugs.METHODS: MTX was covalently linked to insulin. InsulinMTX conjugate was purified by Sephadex G-25 column and analyzed by high performance liquid chromatography.Hepatocellular carcinoma cell membrane fractions were isolated by sucrose density gradient centrifugation.Competitive displacement of 125I-insulin with insulin and insulin-MTX binding to insulin receptors were carried out.Cytoreductive effect of insulin-MTX on human hepatoma BEL7402 cells and human hepatocyte cell line HL7702 was evaluated using the MTT assay.RESULTS: Insulin-MTX competed as effectively as insulin with 125I-insulin for insulin receptors. The values of Kd for insulin-MTX and insulin were 93.82±19.32 nmol/L and 5.01±1.24 nmol/L, respectively. The value of Kd for insulinMTX was significantly increased in comparison with insulin (t=7.2532,n=4, P<0.005). Insulin-MTX inhibited the growth of human hepatoma cells (BEL7402) almost as potently as MTX. The inhibitory effect reached a peak on the 5 th day when the growth of cells was inhibited by 79% at a concentration of 5.0 μg/mL insulin-MTX. Treatment with 5.0 μg/mL of MTX and 5.0 μg/mL of insulin-MTX merely resulted in inhibition of HL7702 cells by 31.5% and 7.8%on the 5 th day.CONCLUSION: Insulin-MTX specifically recognizes insulin receptors and inhibits the growth of BEL7402 cells. These results suggest that insulin can be used as a carrier in receptor mediated carcinoma-targeting therapy.

  5. The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A.

    Science.gov (United States)

    Kounnas, M Z; Morris, R E; Thompson, M R; FitzGerald, D J; Strickland, D K; Saelinger, C B

    1992-06-25

    The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide; this receptor is thought to be responsible for the binding and endocytosis of activated alpha 2-macroglobulin and apoE-enriched beta-very low density lipoprotein. A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE). We demonstrate that the alpha 2 MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable. Furthermore, affinity-purified alpha 2 MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells. The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha 2 MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts. The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the KD measured for the interaction of receptor-associated protein with the purified receptor. Overall, the studies strongly suggest that the alpha 2 MR/LRP is responsible for internalizing PE.

  6. Receptor Binding Ligands to Image Infection

    NARCIS (Netherlands)

    Chianelli, M.; Boerman, O. C.; Malviya, G.; Galli, F.; Oyen, W. J. G.; Signore, A.

    2008-01-01

    The current gold standard for imaging infection is radiolabeled white blood cells. For reasons of safety, simplicity and cost, it would be desirable to have a receptor-specific ligand that could be used for imaging infection and that would allow a differential diagnosis between sterile and septic in

  7. Pirenzepine Promotes the Dimerization of Muscarinic M1 Receptors through a Three-step Binding Process*

    Science.gov (United States)

    Ilien, Brigitte; Glasser, Nicole; Clamme, Jean-Pierre; Didier, Pascal; Piemont, Etienne; Chinnappan, Raja; Daval, Sandrine B.; Galzi, Jean-Luc; Mely, Yves

    2009-01-01

    Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state. PMID:19451648

  8. Pirenzepine promotes the dimerization of muscarinic M1 receptors through a three-step binding process.

    Science.gov (United States)

    Ilien, Brigitte; Glasser, Nicole; Clamme, Jean-Pierre; Didier, Pascal; Piemont, Etienne; Chinnappan, Raja; Daval, Sandrine B; Galzi, Jean-Luc; Mely, Yves

    2009-07-17

    Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state.

  9. Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.

    Science.gov (United States)

    Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi

    2016-05-03

    Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.

  10. Mannan-binding lectin inhibits Candida albicans-induced cellular responses in PMA-activated THP-1 cells through Toll-like receptor 2 and Toll-like receptor 4.

    Directory of Open Access Journals (Sweden)

    Mingyong Wang

    Full Text Available BACKGROUND: Candida albicans (C. albicans, the most common human fungal pathogen, can cause fatal systemic infections under certain circumstances. Mannan-binding lectin (MBL,a member of the collectin family in the C-type lectin superfamily, is an important serum component associated with innate immunity. Toll-like receptors (TLRs are expressed extensively, and have been shown to be involved in C. albicans-induced cellular responses. We first examined whether MBL modulated heat-killed (HK C. albicans-induced cellular responses in phorbol 12-myristate 13-acetate (PMA-activated human THP-1 macrophages. We then investigated the possible mechanisms of its inhibitory effect. METHODOLOGY/PRINCIPAL FINDING: Enzyme-linked immunosorbent assay (ELISA and reverse transcriptasepolymerase chain reaction (RT-PCR analysis showed that MBL at higher concentrations (10-20 µg/ml significantly attenuated C. albicans-induced chemokine (e.g., IL-8 and proinflammatory cytokine (e.g., TNF-α production from PMA-activated THP-1 cells at both protein and mRNA levels. Electrophoretic mobility shift assay (EMSA and Western blot (WB analysis showed that MBL could inhibit C. albicans-induced nuclear factor-κB (NF-κB DNA binding and its translocation in PMA-activated THP-1 cells. MBL could directly bind to PMA-activated THP-1 cells in the presence of Ca(2+, and this binding decreased TLR2 and TLR4 expressions in C. albicans-induced THP-1 macrophages. Furthermore, the binding could be partially inhibited by both anti-TLR2 monoclonal antibody (clone TL2.1 and anti-TLR4 monoclonal antibody (clone HTA125. In addition, co-immunoprecipitation experiments and microtiter wells assay showed that MBL could directly bind to the recombinant soluble form of extracellular TLR2 domain (sTLR2 and sTLR4. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that MBL can affect proinflammatory cytokine and chemokine expressions by modifying C. albicans-/TLR-signaling pathways. This study supports

  11. Beta 2-adrenergic receptors on eosinophils. Binding and functional studies

    Energy Technology Data Exchange (ETDEWEB)

    Yukawa, T.; Ukena, D.; Kroegel, C.; Chanez, P.; Dent, G.; Chung, K.F.; Barnes, P.J. (National Heart and Lung Institute, Brompton Hospital, London (England))

    1990-06-01

    We have studied the binding characteristics and functional effects of beta-adrenoceptors on human and guinea pig eosinophils. We determined the binding of the beta-antagonist radioligand (125I)pindolol (IPIN) to intact eosinophils obtained from the peritoneal cavity of guinea pigs and from blood of patients with eosinophilia. Specific binding was saturable, and Scatchard analysis showed a single binding site with a dissociation constant (Kd) of 24.6 pM and maximal number of binding sites (Bmax) of 7,166 per cell. ICI 118,551, a beta 2-selective antagonist, inhibited IPIN binding with a Ki value of 0.28 nM and was approximately 5,000-fold more effective than the beta 1-selective antagonist, atenolol. Isoproterenol increased cAMP levels about 5.5-fold above basal levels (EC50 = 25 microM); albuterol, a beta 2-agonist, behaved as a partial agonist with a maximal stimulation of 80%. Binding to human eosinophils gave similar results with a Kd of 25.3 pM and a Bmax corresponding to 4,333 sites per cell. Incubation of both human and guinea pig eosinophils with opsonized zymosan (2 mg/ml) or with phorbol myristate acetate (PMA) (10(-8) and 10(-6) M) resulted in superoxide anion generation and the release of eosinophil peroxidase; albuterol (10(-7) to 10(-5) M) had no inhibitory effect on the release of these products. Thus, eosinophils from patients with eosinophilia and from the peritoneal cavity of guinea pigs possess beta-receptors of the beta 2-subtype that are coupled to adenylate cyclase; however, these receptors do not modulate oxidative metabolism or degranulation. The possible therapeutic consequences of these observations to asthma are discussed.

  12. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Varnum, Susan M.

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

  13. Treponema pallidum receptor binding proteins interact with fibronectin

    Energy Technology Data Exchange (ETDEWEB)

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

    1983-06-01

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

  14. Sialyloligosaccharide receptors of binding variants of polyoma virus.

    Science.gov (United States)

    Cahan, L D; Singh, R; Paulson, J C

    1983-10-30

    Hemagglutination and lytic infection of host cells by polyoma virus has been shown to require specific sialyloligosaccharide structures. The nature of the sialyloligosaccharide sequence recognized by three binding variants of polyoma virus, the large plaque (LP), small plaque (SP), and Py235 variants, was examined. Hemagglutination of native erythrocytes and erythrocytes derivatized with highly specific sialyltransferases to contain cell surface sialyloligosaccharides of defined sequence was compared for the three variants. In addition, soluble glycoprotein inhibitors of known sialyloligosaccharide structure were used to further elucidate the specificities of the three variants. There are important differences in the receptors for these variants. While all three appear to bind the structure NeuAc alpha 2,3Gal beta 1,3GalNAc the LP and Py235 variant bind the disialyl structure NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAc with much lower affinity than does the SP virus. It is suggested that polyoma virus adsorption to cells may depend on the cell surface content of at least three different sialyloligosaccharide sequences and the relative abilities of the virus variant to utilize them as receptor determinants.

  15. Estradiol Binds to Insulin and Insulin Receptor Decreasing Insulin Binding in vitro

    Directory of Open Access Journals (Sweden)

    Robert eRoot-Bernstein

    2014-07-01

    Full Text Available Rationale: Insulin resistance associated with hyperestrogenemias occurs in gestational diabetes mellitus, polycystic ovary syndrome, ovarian hyperstimulation syndrome, estrogen therapies, metabolic syndrome and obesity. The mechanism by which insulin and estrogen interact is unknown. We hypothesize that estrogen binds directly to insulin and the insulin receptor producing insulin resistance.Objectives: To determine the binding constants of steroid hormones to insulin, the insulin receptor, and insulin-like peptides derived from the insulin receptor; and to investigate the effect of estrogens on the binding of insulin to its receptor.Methods: Ultraviolet spectroscopy, capillary electrophoresis and NMR demonstrated estrogen binding to insulin and its receptor. Horse-radish peroxidase-linked insulin was used in an ELISA-like procedure to measure the effect of estradiol on binding of insulin to its receptor. Measurements: Binding constants for estrogens to insulin and the insulin receptor were determined by concentration-dependent spectral shifts. The effect of estradiol on insulin-HRP binding to its receptor was determined by shifts in the insulin binding curve. Main Results: Estradiol bound to insulin with a Kd of 12 x 10-9 M and to the insulin receptor with a Kd of 24 x 10-9 M, while other hormones had significantly less affinity. 200 nM estradiol shifted the binding curve of insulin to its receptor 0.8 log units to the right. Conclusions: Estradiol concentrations in many hyperestrogenemic syndromes are sufficient to interfere with insulin binding to its receptor producing significant insulin resistance.

  16. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.; Mushrush, Darren J.; Pruitt, Rory N.; Spiller, Benjamin W.; Barbieri, Joseph T.; Lacy, D. Borden (Vanderbilt); (MCW)

    2010-10-19

    Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

  17. Using Global Analysis to Extend the Accuracy and Precision of Binding Measurements with T cell Receptors and Their Peptide/MHC Ligands

    Science.gov (United States)

    Blevins, Sydney J.; Baker, Brian M.

    2017-01-01

    In cellular immunity, clonally distributed T cell receptors (TCRs) engage complexes of peptides bound to major histocompatibility complex proteins (pMHCs). In the interactions of TCRs with pMHCs, regions of restricted and variable diversity align in a structurally complex fashion. Many studies have used mutagenesis to attempt to understand the “roles” played by various interface components in determining TCR recognition properties such as specificity and cross-reactivity. However, these measurements are often complicated or even compromised by the weak affinities TCRs maintain toward pMHC. Here, we demonstrate how global analysis of multiple datasets can be used to significantly extend the accuracy and precision of such TCR binding experiments. Application of this approach should positively impact efforts to understand TCR recognition and facilitate the creation of mutational databases to help engineer TCRs with tuned molecular recognition properties. We also show how global analysis can be used to analyze double mutant cycles in TCR-pMHC interfaces, which can lead to new insights into immune recognition. PMID:28197404

  18. Differential ligand binding affinities of human estrogen receptor-α isoforms

    OpenAIRE

    Amanda H.Y. Lin; Li, Rachel W. S.; Ho, Eva Y. W.; George P H Leung; Susan W S Leung; Paul M Vanhoutte; Man, Ricky Y K

    2013-01-01

    Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cel...

  19. Binding Mode of Insulin Receptor and Agonist Peptide

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Insulin is a protein hormone secreted by pancreatic β cells. One of its main functions is to keep the balance of glucose inside the body by regulating the absorption and metabolism of glucose in the periphery tissue, as well as the production and storage of hepatic glycogen. The insulin receptor is a transmembrane glycoprotein in which two α subunits with a molecular weight of 135 kD and twoβ subunits with a molecular weight of 95 kD are joined by a disulfide bond to form a β-α-α-β structure. The extracellular α subunit, especially, its three domains near the N-terminal are partially responsible for signal transduction or ligand-binding, as indicated by the experiments. The extracellular α subunits are involved in binding the ligands. The experimental results indicate that the three domains of the N-terminal of the α subunits are the main determinative parts of the insulin receptor to bind the insulin or mimetic peptide.We employed the extracellular domain (PDBID: 1IGR) of the insulin-like growth factor-1 receptor (IGF-1 R ) as the template to simulate and optimize the spatial structures of the three domains in the extracellular domain of the insulin receptor, which includes 468 residues. The work was accomplished by making use of the homology program in the Insight Ⅱ package on an Origin3800 server. The docking calculations of the insulin receptor obtained by homology with hexapeptides were carried out by means of the program Affinity. The analysis indicated that there were hydrogen bonding, and electrostatic and hydrophobic effects in the docking complex of the insulin receptor with hexapeptides.Moreover, we described the spatial orientation of a mimetic peptide with agonist activity in the docking complex. We obtained a rough model of binding of DLAPSQ or STIVYS with the insulin receptor, which provides the powerful theoretical support for designing the minimal insulin mimetic peptide with agonist activity, making it possible to develop oral small

  20. The Autocrine Mitogenic Loop of the Ciliate Euplotes raikovi: The Pheromone Membrane-bound Forms Are the Cell Binding Sites and Potential Signaling Receptors of Soluble Pheromones

    Science.gov (United States)

    Ortenzi, Claudio; Alimenti, Claudio; Vallesi, Adriana; Di Pretoro, Barbara; Terza, Antonietta La; Luporini, Pierangelo

    2000-01-01

    Homologous proteins, denoted pheromones, promote cell mitotic proliferation and mating pair formation in the ciliate Euplotes raikovi, according to whether they bind to cells in an autocrine- or paracrine-like manner. The primary transcripts of the genes encoding these proteins undergo alternate splicing, which generates at least two distinct mRNAs. One is specific for the soluble pheromone, the other for a pheromone isoform that remains anchored to the cell surface as a type II protein, whose extracellular C-terminal region is structurally equivalent to the secreted form. The 15-kDa membrane-bound isoform of pheromone Er-1, denoted Er-1mem and synthesized by the same E. raikovi cells that secrete Er-1, has been purified from cell membranes by affinity chromatography prepared with matrix-bound Er-1, and its extracellular and cytoplasmic regions have been expressed as recombinant proteins. Using the purified material and these recombinant proteins, it has been shown that Er-1mem has the property of binding pheromones competitively through its extracellular pheromone-like domain and associating reversibly and specifically with a guanine nucleotide-binding protein through its intracellular domain. It has been concluded that the membrane-bound pheromone isoforms of E. raikovi represent the cell effective pheromone binding sites and are functionally equipped for transducing the signal generated by this binding. PMID:10749941

  1. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B

    2003-11-01

    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

  2. Soluble and cell surface receptors for tumor necrosis factor

    DEFF Research Database (Denmark)

    Wallach, D; Engelmann, H; Nophar, Y

    1991-01-01

    Tumor necrosis factor (TNF) initiates its multiple effects on cell function by binding at a high affinity to specific cell surface receptors. Two different molecular species of these receptors, which are expressed differentially in different cells, have been identified. The cDNAs of both receptor...... have recently been cloned. Antibodies to one of these receptor species (the p55, type I receptor) can trigger a variety of TNF like effects by cross-linking of the receptor molecules. Thus, it is not TNF itself but its receptors that provide the signal for the response to this cytokine...... in certain pathological situations. Release of the soluble receptors from the cells seems to occur by proteolytic cleavage of the cell surface forms and appears to be a way of down-regulating the cell response to TNF. Because of their ability to bind TNF, the soluble receptors exert an inhibitory effect...

  3. Menthol binding and inhibition of α7-nicotinic acetylcholine receptors.

    Science.gov (United States)

    Ashoor, Abrar; Nordman, Jacob C; Veltri, Daniel; Yang, Keun-Hang Susan; Al Kury, Lina; Shuba, Yaroslav; Mahgoub, Mohamed; Howarth, Frank C; Sadek, Bassem; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-01-01

    Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca(2+)-dependent Cl(-) channels, since menthol inhibition remained unchanged by intracellular injection of the Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing Ba(2+). Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [(125)I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca(2+) transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner.

  4. Menthol binding and inhibition of α7-nicotinic acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Abrar Ashoor

    Full Text Available Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca(2+-dependent Cl(- channels, since menthol inhibition remained unchanged by intracellular injection of the Ca(2+ chelator BAPTA and perfusion with Ca(2+-free bathing solution containing Ba(2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [(125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca(2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner.

  5. Dissociation of FK506-binding protein 12.6 kD from ryanodine receptor in bronchial smooth muscle cells in airway hyperresponsiveness in asthma.

    Science.gov (United States)

    Du, Ying; Zhao, Jianhong; Li, Xi; Jin, Si; Ma, Wan-Li; Mu, Qing; Xu, Shuxiang; Yang, Jie; Rao, Shanshan; Zhu, Liping; Xin, Jianbao; Cai, Peng-Cheng; Su, Yunchao; Ye, Hong

    2014-02-01

    Airway hyperresponsiveness (AHR) in asthma is predominantly caused by increased sensitivity of bronchial smooth muscle cells (BSMCs) to stimuli. The sarcoplasmic reticulum (SR)-Ca(2+) release channel, known as ryanodine receptor (RyR), mediates the contractive response of BSMCs to stimuli. FK506-binding protein 12.6 kD (FKBP12.6) stabilizes the RyR2 channel in a closed state. However, the interaction of FKBP12.6 with RyR2 in AHR remains unknown. This study examined the interaction of FKBP12.6 with RyR2 in BSMCs in AHR of asthma. The interaction of FKBP12.6 with RyR2 and FKBP12.6 expression was determined in a rat asthma model and in BSMCs treated with inflammatory cytokines. The calcium responses to contractile agonists were determined in BSMCs with overexpression and knockdown of FKBP12.6. Asthmatic serum, IL-5, IL-13, and TNF-α enhance the calcium response of BSMCs to contractile agonists and cause dissociation of FKBP12.6 from RyR2 and a decrease in FKBP12.6 gene expression in BSMCs in culture and in ovalbumin (OVA)-sensitized and -challenged rats. Knockdown of FKBP12.6 in BSMCs causes a decrease in the association of RyR2 with FKBP12.6 and an increase in the calcium response of BSMCs. Overexpression of FKBP12.6 increases the association of FKBP12.6 with RyR2, decreases the calcium response of BSMCs, and normalizes airway responsiveness in OVA-sensitized and -challenged rats. Dissociation of FKBP12.6 from RyR2 in BSMCs is responsible for the increased calcium response contributing to AHR in asthma. Manipulating the interaction of FKBP12.6 with RyR2 might be a novel and useful treatment for asthma.

  6. Development of Gamma-Emitting Receptor Binding Radiopharmace

    Energy Technology Data Exchange (ETDEWEB)

    Reba, Richard

    2003-02-20

    The long-term objective is to develop blood-brain barrier (BBB) permeable m2-selective (relative to m1, m3, and m4) receptor-binding radiotracers and utilize these radiotracers for quantifying receptor concentrations obtained from PET or SPECT images of human brain. In initial studies, we concluded that the lipophilicity and high affinity prevented (R,S)-I-QNB from reaching a flow-independent and receptor-dependent state in a reasonable time. Thus, it was clear that (R,S)-I-QNB should be modified. Therefore, during the last portion of this funded research, we proposed that more polar heterocycles should help accomplish that. Since reports of others concluded that radiobromination and radiofluorination of the unactivated phenyl ring is not feasible (Newkome et al,,1982), we, therefore, explored during this grant period a series of analogues of (R)-QNB in which one or both of the six-membered phenyl rings is replaced by a five-membered thienyl (Boulay et al., 1995), or furyl ring. The chemistry specific aims were to synthesize novel compounds designed to be m2-selective mAChR ligands capable of penetrating into the CNS, and develop methods for efficient radiolabeling of promising m2-selective muscarinic ligands. The pharmacology specific aims were to determine the affinity and subtype-selectivity of the novel compounds using competition binding studies with membranes from cells that express each of the five muscarinic receptor subtypes, to determine the ability of the promising non-radioactive compounds and radiolabeled novel compounds to cross the BBB, to determine the biodistribution, in-vivo pharmacokinetics, and in-vitm kinetics of promising m2-selective radioligands and to determine the distribution of receptors for the novel m2-selective radioligands using quantitative autoradiography of rat brain, and compare this distribution to the distribution of known m2-selective compounds.

  7. Malondialdehyde-acetaldehyde haptenated protein binds macrophage scavenger receptor(s) and induces lysosomal damage.

    Science.gov (United States)

    Willis, Monte S; Klassen, Lynell W; Carlson, Deborah L; Brouse, Chad F; Thiele, Geoffrey M

    2004-07-01

    There is evidence that the chemical modification of proteins (haptens) with malondialdehyde-acetaldehyde (MAA) and the immune response to these haptenated proteins is associated with the initiation and/or progression of alcohol liver disease. Experimentally, proteins modified with MAA induce antibody and T cell responses, which are mediated by scavenger receptor(s). Moreover, macrophages have been shown to play an important role in processing and presenting MAA-haptenated proteins in vitro. In vitro, MAA-modified proteins have been shown to induce both apoptosis and necrosis in a dose- and cell-type-dependent manner. Natural ligands modified by oxidative stress, such as oxidized LDL, similarly initiate not only antibody responses, but also cause cell death by disrupting lysosomes after binding to scavenger receptors and internalization. We therefore investigated the binding, internalization, and lysosomal integrity in a macrophage cell line to a MAA-haptenated protein. We demonstrate for the first time that MAA-haptenated proteins are preferentially bound by scavenger receptors on macrophages, which internalize the ligands and shuttle them to lysosomes. Moreover, MAA-haptenated proteins are demonstrated to be associated with a rapid dose-dependent disruption in lysosomal integrity, resulting in leakage and caspase activation. Similarly, as hen egg lysozyme (HEL)-MAA concentrations increased (>31.3 microg/ml), increased levels of apoptosis and a G1/S cell cycle checkpoint inhibition were identified. This study identifies mechanisms by which MAA-haptenated proteins are taken up by a representative antigen-presenting cell and may delineate steps by which MAA-haptenated proteins induce cell death and induce their immunogenicity to the carrier protein. Copyright 2004 Elsevier B.V.

  8. Exploiting Receptor Competition to Enhance Nanoparticle Binding Selectivity

    Science.gov (United States)

    Angioletti-Uberti, Stefano

    2017-02-01

    Nanoparticles functionalized with multiple ligands can be programed to bind biological targets depending on the receptors they express, providing a general mechanism exploited in various technologies, from selective drug delivery to biosensing. For binding to be highly selective, ligands should exclusively interact with specific targeted receptors, because the formation of bonds with other, untargeted ones would lead to nonspecific binding and potentially harmful behavior. This poses a particular problem for multivalent nanoparticles, because even very weak bonds can collectively lead to strong binding. A statistical mechanical model is used here to describe how competition between different receptors together with multivalent effects can be harnessed to design ligand-functionalized nanoparticles insensitive to the presence of untargeted receptors, preventing nonspecific binding.

  9. Dysregulation of Striatal Dopamine Receptor Binding in Suicide.

    Science.gov (United States)

    Fitzgerald, Megan L; Kassir, Suham A; Underwood, Mark D; Bakalian, Mihran J; Mann, J John; Arango, Victoria

    2017-03-01

    Inconsistent evidence implicates disruptions of striatal dopaminergic indices in suicide and major depression. To determine whether there are alterations in the striatal dopamine system in suicide, we conducted a quantitative autoradiographic survey of dopamine transporter (DAT; [(3)H]mazindol), D1 receptor ([(3)H]SCH23390), and D2 receptor ([(3)H]sulpiride) binding in the dorsal striatum postmortem from matched suicides and controls. Axis I and axis II psychiatric diagnosis, recent treatment history, and early life adversity (ELA) were determined by psychological autopsy. Mean DAT, D2, and D1 receptor binding did not differ in suicide. However, there was a positive correlation between D1 and D2 receptor binding in the dorsal striatum of control subjects (R(2)=0.31, pELA, there was no correlation between striatal DAT and D1 receptor binding (R(2)=0.07, p=0.33), although DAT and D1 receptor binding was positively correlated in subjects with no report of ELA (R(2)=0.32, pELA-related mean differences. Binding of D1 receptors and DAT throughout the striatum correlated negatively with age (D1 receptor: R(2)=0.12, pELA or age.

  10. Hierarchy of ADAM12 binding to integrins in tumor cells

    DEFF Research Database (Denmark)

    Thodeti, Charles Kumar; Fröhlich, Camilla; Nielsen, Christian Kamp

    2005-01-01

    ADAMs (a disintegrin and metalloprotease) comprise a family of cell surface proteins with protease and cell-binding activities. Using different forms and fragments of ADAM12 as substrates in cell adhesion and spreading assays, we demonstrated that alpha9beta1 integrin is the main receptor for ADA...

  11. THE RECEPTOR BINDING AFFINITIES, ANTIPROGESTERONE AND ANTIGLUCOCORTICOID ACTIVITIES OF MIFEPRISTONE AND LILOPRISTONE

    Institute of Scientific and Technical Information of China (English)

    LIUYong-Qiang; WUXi-Rui

    1989-01-01

    With radioligand binding assays, the receptor binding affmities of mifepristone and lilopristone to the rabbit uterus cytosol progesterone receptor and the rat fiver cytosol glucocorticoid receptor have been measured. The relative binding affinities ( RBA ) of

  12. A peptide motif from the second fibronectin module of the neural cell adhesion molecule, NCAM, NLIKQDDGGSPIRHY, is a binding site for the FGF receptor

    DEFF Research Database (Denmark)

    Jacobsen, Jacob Hedemand; Kiselyov, Vladislav; Bock, Elisabeth

    2008-01-01

    The mechanism of fibroblast growth factor receptor (FGFR) activation by the neural cell adhesion molecule (NCAM) is not well understood. A motif in the second NCAM fibronectin type III (FN3) module, termed FGL, has by means of nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) a...

  13. Identification of different binding sites in the dendritic cell-specific receptor DC-SIGN for intercellular adhesion molecule 3 and HIV-1.

    NARCIS (Netherlands)

    Geijtenbeek, T.B.; Duijnhoven, G.C.F. van; Vliet, S. van; Krieger, E.; Vriend, G.; Figdor, C.G.; Kooyk, Y. van

    2002-01-01

    The novel dendritic cell (DC)-specific human immunodeficiency virus type 1 (HIV-1) receptor DC-SIGN plays a key role in the dissemination of HIV-1 by DC. DC-SIGN is thought to capture HIV-1 at mucosal sites of entry, facilitating transport to lymphoid tissues, where DC-SIGN efficiently transmits HIV

  14. Identification of different binding sites in the dendritic cell-specific receptor DC-SIGN for intercellular adhesion molecule 3 and HIV-1.

    NARCIS (Netherlands)

    Geijtenbeek, T.B.; Duijnhoven, G.C.F. van; Vliet, S. van; Krieger, E.; Vriend, G.; Figdor, C.G.; Kooyk, Y. van

    2002-01-01

    The novel dendritic cell (DC)-specific human immunodeficiency virus type 1 (HIV-1) receptor DC-SIGN plays a key role in the dissemination of HIV-1 by DC. DC-SIGN is thought to capture HIV-1 at mucosal sites of entry, facilitating transport to lymphoid tissues, where DC-SIGN efficiently transmits HIV

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

    Science.gov (United States)

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

    2002-03-01

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

  16. Properties of Opiate-Receptor Binding in Rat Brain

    Science.gov (United States)

    Pert, Candace B.; Snyder, Solomon H.

    1973-01-01

    [3H]Naloxone, a potent opiate antagonist, binds stereospecifically to opiate-receptor sites in rat-brain tissue. The binding is time, temperature, and pH dependent and saturable with respect to [3H]naloxone and tissue concentration. The [3H]naloxone-receptor complex formation is bimolecular with a dissociation constant of 20 nM. 15 Opiate agonists and antagonists compete for the same receptors, whose density is 30 pmol/g. Potencies of opiates and their antagonists in displacing [3H]naloxone binding parallel their pharmacological potencies. PMID:4525427

  17. Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities.

    Science.gov (United States)

    Chung, Shan; Quarmby, Valerie; Gao, Xiaoying; Ying, Yong; Lin, Linda; Reed, Chae; Fong, Chris; Lau, Wendy; Qiu, Zhihua J; Shen, Amy; Vanderlaan, Martin; Song, An

    2012-01-01

    The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its "regular" fucosylated counterpart and a series of mixtures containing varying proportions of "regular" and afucosylated materials. Compared with the "regular" fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC 50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.

  18. Structural Basis for Negative Cooperativity in Growth Factor Binding to an EGF Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Alvarado, Diego; Klein, Daryl E.; Lemmon, Mark A. (UPENN-MED)

    2010-09-27

    Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the high-affinity and low-affinity classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

  19. A Cinnamon-Derived Procyanidin Compound Displays Anti-HIV-1 Activity by Blocking Heparan Sulfate- and Co-Receptor- Binding Sites on gp120 and Reverses T Cell Exhaustion via Impeding Tim-3 and PD-1 Upregulation

    Science.gov (United States)

    Connell, Bridgette Janine; Chang, Sui-Yuan; Prakash, Ekambaranellore; Yousfi, Rahima; Mohan, Viswaraman; Posch, Wilfried; Wilflingseder, Doris; Moog, Christiane; Kodama, Eiichi N.; Clayette, Pascal; Lortat-Jacob, Hugues

    2016-01-01

    Amongst the many strategies aiming at inhibiting HIV-1 infection, blocking viral entry has been recently recognized as a very promising approach. Using diverse in vitro models and a broad range of HIV-1 primary patient isolates, we report here that IND02, a type A procyanidin polyphenol extracted from cinnamon, that features trimeric and pentameric forms displays an anti-HIV-1 activity against CXCR4 and CCR5 viruses with 1–7 μM ED50 for the trimer. Competition experiments, using a surface plasmon resonance-based binding assay, revealed that IND02 inhibited envelope binding to CD4 and heparan sulphate (HS) as well as to an antibody (mAb 17b) directed against the gp120 co-receptor binding site with an IC50 in the low μM range. IND02 has thus the remarkable property of simultaneously blocking gp120 binding to its major host cell surface counterparts. Additionally, the IND02-trimer impeded up-regulation of the inhibitory receptors Tim-3 and PD-1 on CD4+ and CD8+ cells, thereby demonstrating its beneficial effect by limiting T cell exhaustion. Among naturally derived products significantly inhibiting HIV-1, the IND02-trimer is the first component demonstrating an entry inhibition property through binding to the viral envelope glycoprotein. These data suggest that cinnamon, a widely consumed spice, could represent a novel and promising candidate for a cost-effective, natural entry inhibitor for HIV-1 which can also down-modulate T cell exhaustion markers Tim-3 and PD-1. PMID:27788205

  20. Rifampicin Inhibits the LPS-induced Expression of Toll-like Receptor 2 via the Suppression of NF-kappaB DNA-binding Activity in RAW 264.7 Cells.

    Science.gov (United States)

    Kim, Seong Keun; Kim, Young Mi; Yeum, Chung Eun; Jin, Song-Hyo; Chae, Gue Tae; Lee, Seong-Beom

    2009-12-01

    Rifampicin is a macrocyclic antibiotic which is used extensively for treatment against Mycobacterium tuberculosis and other mycobacterial infections. Recently, a number of studies have focused on the immune-regulatory effects of rifampicin. Therefore, we hypothesized that rifampicin may influence the TLR2 expression in LPS-activated RAW 264.7 cells. In this study, we determined that rifampicin suppresses LPS-induced TLR2 mRNA expression. The down-regulation of TLR2 expression coincided with decreased production of TNF-alpha. Since NF-kappaB is a major transcription factor that regulates genes for TLR2 and TNF-alpha, we examined the effect of rifampicin on the LPS-induced NF-kappaB activation. Rifampicin inhibited NF-kappaB DNA-binding activity in LPS-activated RAW 264.7 cells, while it did not affect IKKalpha/beta activity. However, rifampicin slightly inhibited the nuclear translocation of NF-kappaB p65. In addition, rifampicin increased physical interaction between pregnane X receptor, a receptor for rifampicin, and NF-kappaB p65, suggesting pregnane X receptor interferes with NF-kappaB binding to DNA. Taken together, our results demonstrate that rifampicin inhibits LPS-induced TLR2 expression, at least in part, via the suppression of NF-kappaB DNA-binding activity in RAW 264.7 cells. Thus, the present results suggest that the rifampicin-mediated inhibition of TLR2 via the suppression of NF-kappaB DNA-binding activity may be a novel mechanism of the immune-suppressive effects of rifampicin.

  1. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

    Science.gov (United States)

    Hardison, D. Ransom; Holland, William C.; McCall, Jennifer R.; Bourdelais, Andrea J.; Baden, Daniel G.; Darius, H. Taiana; Chinain, Mireille; Tester, Patricia A.; Shea, Damian; Flores Quintana, Harold A.; Morris, James A.; Litaker, R. Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®- PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®- PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  2. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    Science.gov (United States)

    Hardison, D Ransom; Holland, William C; McCall, Jennifer R; Bourdelais, Andrea J; Baden, Daniel G; Darius, H Taiana; Chinain, Mireille; Tester, Patricia A; Shea, Damian; Quintana, Harold A Flores; Morris, James A; Litaker, R Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  3. Herpesvirus saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor.

    Science.gov (United States)

    Yao, Zhengbin; Fanslow, William C; Seldin, Michael F; Rousseau, Anne-Marie; Painter, Sally L; Comeau, Michael R; Cohen, Jeffrey I; Spriggs, Melanie K

    2011-11-01

    Herpesvirus Saimiri gene 13 (HVS13) exhibits 57% identity with the predicted sequence of a T cell-derived molecule termed CTLA8. Recombinant HVS13 and CTLA8 stimulate transcriptional factor NF-kappaB activity and Interleukin-6 (IL-6) secretion in fibroblasts, and costimulate T cell proliferation. An HVS13.Fc fusion protein was used to isolate a cDNA encoding a novel receptor that also binds CTLA8. This receptor is unrelated to previously identified cytokine receptor families. A recombinant soluble receptor inhibited T cell proliferation and IL-2 production induced by PHA, concanavalin A (conA), and anti-TCR MAb. These results define CTLA8 and HVS13 as novel cytokines that bind to a novel cytokine receptor. We propose to call these molecules IL-17, vIL-17, and IL-17R, respectively.

  4. Receptor-binding radiotracers: a class of potential radiopharmaceuticals

    Energy Technology Data Exchange (ETDEWEB)

    Eckelman, W.C.; Reba, R.C.; Gibson, R.E.; Rzeszotarski, W.J.; Vieras, F.; Mazaitis, J.K.; Francis, B.

    1979-04-01

    To date no radiopharmaceutical is routinely used to study changes in receptor concentration. Frequently changes in receptor concentration, or the appearance of receptors in tumors, indicates a specific pathologic state. With a receptor-binding radiotracer, in vivo studies of these changes will be possible. A reversible bimolecular model and in vitro tests were used to determine equilibrium constants and maximal target-to-blood ratios for new derivatives. Theoretical calculations showed that derivatives binding to the estrogen receptor, the beta adrenoceptor, or the cholinergic receptor are capable of achieving satisfactory target-to-blood ratios. Using in vitro tests, the apparent affinity constant was determined for five iodinated estrogen derivatives and five derivatives of beta blockers. Results of the in vitro study with derivatives of beta blockers. Results of the in vitro study with derivatives of beta blockers, and in vivo displacement studies using propranolol, indicated that the high heart-to-blood ratios (5 to 20) obtained with the new derivatives were not the result of a specific interaction with the receptor. In this instance factors other than receptor binding control the in vivo distribution. The in vitro assay using estrogen receptors showed that of the five derivatives, iodohexestrol and 17-alpha-iodoethynylestradiol bind to the receptor with the highest affinity. In vivo studies confirmed these results; iodohexestrol gave a uterus-to-blood ratio of 10 in immature rats when plasma-protein binding was blocked. With a tritiated muscarinic cholinergic blocking agent, heart-to-blood ratios near the theoretical maximum were obtained. This compound most closely follows the mechanism described by the model. Use of the theoretical model in conjunction with in vitro assays can greatly aid in the design of this new class of receptor-binding radiopharmaceuticals.

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

    Science.gov (United States)

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

    2000-03-01

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

  6. Activation of a GTP-binding protein and a GTP-binding-protein-coupled receptor kinase (beta-adrenergic-receptor kinase-1) by a muscarinic receptor m2 mutant lacking phosphorylation sites.

    Science.gov (United States)

    Kameyama, K; Haga, K; Haga, T; Moro, O; Sadée, W

    1994-12-01

    A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(o) and G(i)2, displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [3H]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[35S]thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]quinuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.

  7. Egf binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3.

    OpenAIRE

    King, C. R.; Borrello, I; Bellot, F; Comoglio, P; Schlessinger, J

    1988-01-01

    The epidermal growth factor receptor (EGF-R) and the erbB-2 proto-oncogene product protein are closely related by their structural homology and their shared enzymatic activity as autophosphorylating tyrosine kinases. We show that in mammary tumor cells (SK-BR-3) EGF causes a rapid increase in tyrosine phosphorylation of the erbB-2 protein. Phosphorylation of erbB-2 does not occur in cells lacking the EGF-R (MDA-MB-453). Phosphorylation of erbB-2 in SK-BR-3 cells is blocked if EGF is prevented...

  8. Effects of the binding of a dextran derivative on fibroblast growth factor 2: secondary structure and receptor-binding studies.

    Science.gov (United States)

    Bittoun, P; Bagheri-Yarmand, R; Chaubet, F; Crépin, M; Jozefonvicz, J; Fermandjian, S

    1999-06-15

    CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.

  9. Preliminary Molecular Dynamic Simulations of the Estrogen Receptor Alpha Ligand Binding Domain from Antagonist to Apo

    Directory of Open Access Journals (Sweden)

    Adrian E. Roitberg

    2008-06-01

    Full Text Available Estrogen receptors (ER are known as nuclear receptors. They exist in the cytoplasm of human cells and serves as a DNA binding transcription factor that regulates gene expression. However the estrogen receptor also has additional functions independent of DNA binding. The human estrogen receptor comes in two forms, alpha and beta. This work focuses on the alpha form of the estrogen receptor. The ERα is found in breast cancer cells, ovarian stroma cells, endometrium, and the hypothalamus. It has been suggested that exposure to DDE, a metabolite of DDT, and other pesticides causes conformational changes in the estrogen receptor. Before examining these factors, this work examines the protein unfolding from the antagonist form found in the 3ERT PDB crystal structure. The 3ERT PDB crystal structure has the estrogen receptor bound to the cancer drug 4-hydroxytamoxifen. The 4-hydroxytamoxifen ligand was extracted before the simulation, resulting in new conformational freedom due to absence of van der Waals contacts between the ligand and the receptor. The conformational changes that result expose the binding clef of the co peptide beside Helix 12 of the receptor forming an apo conformation. Two key conformations in the loops at either end of the H12 are produced resulting in the antagonist to apo conformation transformation. The results were produced over a 42ns Molecular Dynamics simulation using the AMBER FF99SB force field.

  10. Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors

    Science.gov (United States)

    Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.

    1990-12-01

    Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.

  11. Binding characteristics of the muscarinic receptor subtype in rabbit pancreas

    Energy Technology Data Exchange (ETDEWEB)

    van Zwam, A.J.; Willems, P.H.; Rodrigues de Miranda, J.F.; de Pont, J.J.; van Ginneken, C.A. (Catholic Univ. of Nijmegen (Netherlands))

    1990-01-01

    The muscarinic receptor in the rabbit pancreas was characterized with the use of the labeled ligand ({sup 3}H)-(-)-quinuclidinyl-benzylate (({sup 3}H)-(-)-QNB). Specific binding of ({sup 3}H)-(-)-QNB to pancreatic acini was found to be reversible and of high affinity, with an equilibrium dissociation constant (KD) of 68 pmol/l and a receptor density (RT) of 170 fmol/mg protein. Agonist binding behaviour was investigated by displacement of ({sup 3}H)-(-)-QNB binding by eight agonists like arecoline, arecadine-propargylester (APE) and carbachol, yielding only low affinity binding sites. The inhibition of ({sup 3}H)-(-)-QNB binding by the selective antagonists pirenzepine, hexahydrosiladifenidol (HHSiD) and (11-(2-(diethyl-amino)-methyl-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyr ido (2,3-b) (1,4) benzodiazepin-6-one (AF-DX 116) confirmed the M3 nature of the rabbit pancreatic receptor.

  12. Sex Differences in Serotonin 1 Receptor Binding in Rat Brain

    Science.gov (United States)

    Fischette, Christine T.; Biegon, Anat; McEwen, Bruce S.

    1983-10-01

    Male and female rats exhibit sex differences in binding by serotonin 1 receptors in discrete areas of the brain, some of which have been implicated in the control of ovulation and of gonadotropin release. The sex-specific changes in binding, which occur in response to the same hormonal (estrogenic) stimulus, are due to changes in the number of binding sites. Castration alone also affects the number of binding sites in certain areas. The results lead to the conclusion that peripheral hormones modulate binding by serotonin 1 receptors. The status of the serotonin receptor system may affect the reproductive capacity of an organism and may be related to sex-linked emotional disturbances in humans.

  13. Staphylococcal superantigen-like protein 3 binds to the Toll-like receptor 2 extracellular domain and inhibits cytokine production induced by Staphylococcus aureus, cell wall component, or lipopeptides in murine macrophages.

    Science.gov (United States)

    Yokoyama, Ryosuke; Itoh, Saotomo; Kamoshida, Go; Takii, Takemasa; Fujii, Satoshi; Tsuji, Tsutomu; Onozaki, Kikuo

    2012-08-01

    Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins sharing structural similarity with superantigens, but no superantigenic activity. Corresponding host target proteins or receptors against a portion of SSLs in the family have been identified. In this study, we show that SSL3 specifically binds to Toll-like receptor 2 (TLR2) and inhibits the stimulation of macrophages by TLR2 ligands. An approximately 100-kDa protein was recovered by using recombinant His-tagged SSL3-conjugated Sepharose from the lysate of porcine spleen, and the protein was identified as porcine TLR2 by peptide mass fingerprinting analysis. The SSL3-conjugated Sepharose recovered human and mouse TLR2 but not TLR4 from human neutrophils and mouse macrophage RAW 264.7 cells, as well as a recombinant TLR2 extracellular domain chimera protein. The production levels of interleukin 12 (IL-12) from mouse macrophages treated with heat-killed Staphylococcus aureus and of tumor necrosis factor alpha (TNF-α) from RAW 264.7 cells induced by peptidoglycan or lipopeptide TLR2 ligands were strongly suppressed in the presence of SSL3. The mutation of consensus sialic acid-containing glycan-binding residues in SSL3 did not abrogate the binding ability to TLR2 or inhibitory activity on TLR2, indicating that the interaction of SSL3 with TLR2 was independent of the sialic acid-containing glycan-binding residues. These findings demonstrate that SSL3 is able to bind the extracellular domain of TLR2 and interfere with TLR2 function. The present study provides a novel mechanism of SSL3 in immune evasion of S. aureus via interfering with its recognition by innate immune cells.

  14. Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4

    Institute of Scientific and Technical Information of China (English)

    Nianshuang Wang; Xuanling Shi; Liwei Jiang; Senyan Zhang; Dongli Wang; Pei Tong; Dongxing Guo

    2013-01-01

    The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor,dipeptidyl peptidase 4 (DPP4).Sequence comparison and modeling analysis have revealed a putative receptor-binding domain (RBD) on the viral spike,which mediates this interaction.We report the 3.0 (A)resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4.Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain.The receptor-binding subdomain interacts with DPP4 p-propeller but not its intrinsic hydrolase domain.MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains,but are notably divergent in the receptorbinding subdomain.Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into the target cell.The atomic details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction,which can guide development of therapeutics and vaccines against MERS-CoV infection.

  15. Receptor binding sites for atrial natriuretic factor are expressed by brown adipose tissue

    Energy Technology Data Exchange (ETDEWEB)

    Bacay, A.C.; Mantyh, C.R.; Vigna, S.R.; Mantyh, P.W. (Wadsworth VA Medical Center, Los Angeles, CA (USA))

    1988-09-01

    To explore the possibility that atrial natriuretic factor (ANF) is involved in thermoregulation we used quantitative receptor autoradiography and homogenate receptor binding assays to identify ANF bindings sites in neonatal rat and sheep brown adipose tissue, respectively. Using quantitative receptor autoradiography were were able to localize high levels of specific binding sites for {sup 125}I-rat ANF in neonatal rat brown adipose tissue. Homogenate binding assays on sheep brown fat demonstrated that the radioligand was binding to the membrane fraction and that the specific binding was not due to a lipophilic interaction between {sup 125}I-rat ANF and brown fat. Specific binding of {sup 125}I-rat ANF to the membranes of brown fat cells was inhibited by unlabeled rat ANF with a Ki of 8.0 x 10(-9) M, but not by unrelated peptides. These studies demonstrate that brown fat cells express high levels of ANF receptor binding sites in neonatal rat and sheep and suggest that ANF may play a role in thermoregulation.

  16. Binding characteristics of prostaglandin E sub 2 receptor in submandibular glands: Effect of ethanol

    Energy Technology Data Exchange (ETDEWEB)

    Wuwang, C.Y.; Lim, C.; Yao, P.; Wang, S.L.; Slomiany, A.; Slomiany, B.L. (UMDNJ, Newark, NJ (United States))

    1991-03-11

    Prostaglandin (PG) of the E series are known to stimulate saliva flow and mucin secretion in salivary glands, however, the cellular mechanism of this action remains unclear. Binding of PGE to specific binding site may be the initial step in the sequence of events that result in various biological activities. The authors first characterized PGE{sub 2} receptor binding in rat submandibular glandmembranes. The binding was specific and reversible. Scatchard analysis demonstrated that the receptor consists of two binding sites. Since ethanol has been reported to diminish salivary secretion, they further investigated whether this detrimental effect was due to the alteration of PGE receptor. Submandibular glands were dissected from rats, minced, suspended in DMEM and incubated at 37C for 2 hr under 95% O{sub 2}-5% CO{sub 2} in the absence or presence of various concentrations of ethanol. After incubation, cell membranes were prepared and receptor binding assayed. The results indicated that ethanol caused an increase in PGE{sub 2} receptor binding. The specific binding increased by 30% at 2.5% ethanol and by 50% at 5% ethanol. Scatchard analysis of 5% ethanol-treated samples indicated that ethanol-induced increase of PGE{sub 2} binding was due to a 35% decrease and a 2.3-fold decrease of Kds of the high and low affinity receptor, respectively. The binding capacities were not changed by ethanol. It is suggested that ethanol causes an up-regulation of PGE{sub 2} receptor in submandibular glands.

  17. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C. (Scripps); (UW)

    2011-11-02

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  18. Genetic, functional and molecular features of glucocorticoid receptor binding.

    Directory of Open Access Journals (Sweden)

    Francesca Luca

    Full Text Available Glucocorticoids (GCs are key mediators of stress response and are widely used as pharmacological agents to treat immune diseases, such as asthma and inflammatory bowel disease, and certain types of cancer. GCs act mainly by activating the GC receptor (GR, which interacts with other transcription factors to regulate gene expression. Here, we combined different functional genomics approaches to gain molecular insights into the mechanisms of action of GC. By profiling the transcriptional response to GC over time in 4 Yoruba (YRI and 4 Tuscans (TSI lymphoblastoid cell lines (LCLs, we suggest that the transcriptional response to GC is variable not only in time, but also in direction (positive or negative depending on the presence of specific interacting transcription factors. Accordingly, when we performed ChIP-seq for GR and NF-κB in two YRI LCLs treated with GC or with vehicle control, we observed that features of GR binding sites differ for up- and down-regulated genes. Finally, we show that eQTLs that affect expression patterns only in the presence of GC are 1.9-fold more likely to occur in GR binding sites, compared to eQTLs that affect expression only in its absence. Our results indicate that genetic variation at GR and interacting transcription factors binding sites influences variability in gene expression, and attest to the power of combining different functional genomic approaches.

  19. Risperidone treatment increases CB1 receptor binding in rat brain

    DEFF Research Database (Denmark)

    Secher, Anna; Husum, Henriette; Holst, Birgitte

    2010-01-01

    BACKGROUND/AIMS: Body weight gain is a common side effect of treatment with antipsychotics, but the mechanisms underlying this weight gain are unknown. Several factors may be involved in antipsychotic-induced body weight gain including the cannabinoid receptor 1 (CB(1)), the serotonin receptor 2C...... positively correlated with visceral fat mass. Risperidone treatment increased CB(1) receptor binding in the arcuate nucleus (40%), hippocampus (25-30%) and amygdala (35%) without concurrent alterations in the CB(1) receptor mRNA. Risperidone treatment increased adiponectin mRNA. CONCLUSION: The present study...... showed that risperidone treatment altered CB(1) receptor binding in the rat brain. Risperidone-induced adiposity and metabolic dysfunction in the clinic may be explained by increased CB(1) receptor density in brain regions involved in appetite and regulation of metabolic function....

  20. Noncovalent Interactions within a Synthetic Receptor Can Reinforce Guest Binding

    NARCIS (Netherlands)

    Rodriguez-Docampo, Zaida; Pascu, Sofia I.; Kubik, Stefan; Otto, Sijbren

    2006-01-01

    Structural and thermodynamic data are presented on the binding properties of anion receptors containing two covalently linked cyclopeptide subunits that bind sulfate and iodide anions with micromolar affinity in aqueous solution. A synchrotron X-ray crystal structure of the sulfate complex of one

  1. Pneumocystis carinii glycoprotein A binds macrophage mannose receptors.

    OpenAIRE

    O'Riordan, D.M.; Standing, J E; Limper, A H

    1995-01-01

    Pneumocystis carinii causes life-threatening pneumonia in patients with impaired immunity. Recent studies suggest that alveolar macrophages interact with P. carinii through macrophage mannose receptors. However, the ligand(s) on P. carinii that is recognized by these receptors has not been fully defined. P. carinii contains a major mannose-rich surface antigen complex termed glycoprotein A (gpA). It was therefore hypothesized that gpA binds directly to macrophage mannose receptors and mediate...

  2. Receptor-recognized α₂-macroglobulin binds to cell surface-associated GRP78 and activates mTORC1 and mTORC2 signaling in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Uma K Misra

    Full Text Available OBJECTIVE: Tetrameric α(2-macroglobulin (α(2M, a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α(2M (α(2M* binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α(2M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells. METHODS: Employing immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies. RESULTS: Stimulation of cells with α(2M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, Akt(T308, and Akt(S473 in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α(2M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α(2M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α(2M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of Akt(S473 phosphorylation and levels of p-Akt(S473 in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α(2M*-induced phosphorylation of Akt(S473 phosphorylation in Rictor immunoprecipitates. CONCLUSION: Binding of α(2M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    clusters 2 and 4, PrP(C) and PrP(Sc) fibrils bind only to receptor cluster 4. PrP(Sc) fibrils out-compete PrP(C) for internalization. When endocytosed, PrP(Sc) fibrils are routed to lysosomes, rather than recycled to the cell surface with PrP(C). Thus, although LRP1 binds both forms of PrP, it traffics...

  4. Thyroid hormone receptors bind to defined regions of the growth hormone and placental lactogen genes.

    Science.gov (United States)

    Barlow, J W; Voz, M L; Eliard, P H; Mathy-Harter, M; De Nayer, P; Economidis, I V; Belayew, A; Martial, J A; Rousseau, G G

    1986-12-01

    The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that the thyroid hormone-receptor complex interacts with defined regions of this gene has been investigated in a cell-free system. Nuclear extracts from human lymphoblastoid IM-9 cells containing thyroid hormone receptors were incubated with L-3,5,3'-tri[125I]iodothyronine and calf thymus DNA-cellulose. Restriction fragments of the human growth hormone gene were added to determine their ability to inhibit labeled receptor binding to DNA-cellulose. These fragments encompassed nucleotide sequences from about three kilobase pairs upstream to about four kilobase pairs downstream from the transcription initiation site. The thyroid hormone-receptor complex bound preferentially to the 5'-flanking sequences of the growth hormone gene in a region between nucleotide coordinates -290 and -129. The receptor also bound to an analogous promoter region in the human placental lactogen gene, which has 92% nucleotide sequence homology with the growth hormone gene. These binding regions appear to be distinct from those that are recognized by the receptor for glucocorticoids, which stimulate growth hormone gene expression synergistically with thyroid hormone. The presence of thyroid hormone was required for binding of its receptor to the growth hormone gene promoter, suggesting that thyroid hormone renders the receptor capable of recognizing specific gene regions.

  5. Binding of tropane alkaloids to nicotinic and muscarinic acetylcholine receptors.

    Science.gov (United States)

    Schmeller, T; Sporer, F; Sauerwein, M; Wink, M

    1995-07-01

    Fourteen tropane and related alkaloids were analyzed for their affinity for nicotinic and/or muscarinic acetylcholine receptors. The biogenetic intermediates littorine, 6 beta-hydroxyhyoscyamine, 7 beta-hydroxyhyoscyamine exhibit similar affinities at the muscarinic receptor as scopolamine and atropine. The quarternary derivatives N-methylatropine and N-methylscopolamine show the highest binding with IC50 values of less than 100 pM and 300 pM, respectively. The tropane alkaloids (including cocaine) also bind to the nicotinic acetylcholine receptor, albeit with much lower affinities.

  6. Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

    Science.gov (United States)

    Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

    2013-09-17

    Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

  7. Effect of desipramine on dopamine receptor binding in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Suhara, Tetsuya (National Institute of Radiological Sciences, Chiba (Japan) Jikei Univ., Tokyo (Japan)); Inoue, Osamu; Kobayasi, Kaoru (National Institute of Radiological Sciences, Chiba (Japan))

    1990-01-01

    Effect of desipramine on the in vivo binding of {sup 3}H-SCH23390 and {sup 3}H-N-methylspiperone ({sup 3}H-NMSP) in mouse striatum was studied. The ratio of radioactivity in the striatum to that in the cerebellum at 15 min after i.v. injection of {sup 3}H-SCH23390 or 45 min after injection of {sup 3}H-NMSP were used as indices of dopamine D1 or D2 receptor binding in vivo, respectively. In vivo binding of D1 and D2 receptors was decreased in a dose-dependent manner by acute treatment with desipramine (DMI). A saturation experiment suggested that the DMI-induced reduction in the binding was mainly due to the decrease in the affinity of both receptors. No direct interactions between the dopamine receptors and DMI were observed in vitro by the addition of 1 mM of DMI into striatal homogenate. Other antidepressants such as imipramine, clomipramine, maprotiline and mianserin also decreased the binding of dopamine D1 and D2 receptors. The results indicated an important role of dopamine receptors in the pharmacological effect of antidepressants.

  8. Differential ligand binding affinities of human estrogen receptor-α isoforms.

    Directory of Open Access Journals (Sweden)

    Amanda H Y Lin

    Full Text Available Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66 and the truncated isoforms, estrogen receptor-α46 (ER46 and estrogen receptor-α36 (ER36. However, the binding affinities of the membrane estrogen receptors (mERs remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.

  9. Selenium-binding lactoferrin is taken into corneal epithelial cells by a receptor and prevents corneal damage in dry eye model animals.

    Science.gov (United States)

    Higuchi, Akihiro; Inoue, Hiroyoshi; Kaneko, Yoshio; Oonishi, Erina; Tsubota, Kazuo

    2016-11-11

    The ocular surface is strongly affected by oxidative stress, which causes many ocular diseases including dry eye. Previously, we showed that selenium compounds, e.g., selenoprotein P and Se-lactoferrin, were candidates for treatment of dry eye. This paper shows the efficacy of Se-lactoferrin for the treatment of dry eye compared with Diquas as a control drug using two dry eye models and incorporation of lactoferrin into corneal epithelial cells via lactoferrin receptors. We show the efficacy of Se-lactoferrin eye drops in the tobacco smoke exposure rat dry eye model and short-term rabbit dry eye model, although Diquas eye drops were only effective in the short-term rabbit dry eye model. These results indicate that Se-lactoferrin was useful in the oxidative stress-causing dry eye model. Se-lactoferrin was taken into corneal epithelium cells via lactoferrin receptors. We identified LRP1 as the lactoferrin receptor in the corneal epithelium involved in lactoferrin uptake. Se-lactoferrin eye drops did not irritate the ocular surface of rabbits. Se-lactoferrin was an excellent candidate for treatment of dry eye, reducing oxidative stress by a novel mechanism.

  10. Investigation of in vitro Opioid Receptor Binding Activities of Some Turkish Salvia species

    Directory of Open Access Journals (Sweden)

    Özge Gündüz Çınar

    2011-01-01

    Full Text Available Kappa Opioid Peptide Receptor (KOPr activation produces analgesic, psychotomimetic, diuretic and antipruritic effects. KOPr ligands are investigated for their potential roles in the treatment of addiction, depression, feeding behavior, psychosis and schizophrenia. In this study the methanolic extracts of a number of Salvia species which are native to Turkey (S. tomentosa, S. tchihatcheffii , S. rosifolia, S. dichroantha and S. sclarea were tested for their potential binding to opioid receptors in rat brain membranes and Chinese Hamster Ovary Cells expressing human KOPr (CHO-KOPh. [ 3H]Diprenorphine, an unselective opioid antagonist, was utilized in the radioligand receptor binding assays. All extracts (0.11 mg/ml inhibited the [ 3H]Diprenorphine binding with ranging KOPr binding affinities. More than 50% inhibition of diprenorphine binding was shown only with Salvia dichroantha and Salvia sclarea both in rat brain membranes and CHO-KOPh membranes.Among them Salvia sclarea deserves further investigation for its active component(s and its pharmacological characterization. This study clearly demonstrates the potential opioid receptor binding activities of several Turkish Salvia species. This work constitutes the first study on in vitro opioid receptor binding activities of Salvia species from the Turkish flora.

  11. Nanomechanical mapping of first binding steps of a virus to animal cells

    Science.gov (United States)

    Alsteens, David; Newton, Richard; Schubert, Rajib; Martinez-Martin, David; Delguste, Martin; Roska, Botond; Müller, Daniel J.

    2017-02-01

    Viral infection is initiated when a virus binds to cell surface receptors. Because the cell membrane is dynamic and heterogeneous, imaging living cells and simultaneously quantifying the first viral binding events is difficult. Here, we show an atomic force and confocal microscopy set-up that allows the surface receptor landscape of cells to be imaged and the virus binding events within the first millisecond of contact with the cell to be mapped at high resolution (cell surface receptors. We find that the first bond formed between the viral glycoprotein and its cognate cell surface receptor has relatively low lifetime and free energy, but this increases as additional bonds form rapidly (≤1 ms). The formation of additional bonds occurs with positive allosteric modulation and the three binding sites of the viral glycoprotein are quickly occupied. Our quantitative approach can be readily applied to study the binding of other viruses to animal cells.

  12. Nanomechanical mapping of first binding steps of a virus to animal cells

    Science.gov (United States)

    Alsteens, David; Newton, Richard; Schubert, Rajib; Martinez-Martin, David; Delguste, Martin; Roska, Botond; Müller, Daniel J.

    2016-10-01

    Viral infection is initiated when a virus binds to cell surface receptors. Because the cell membrane is dynamic and heterogeneous, imaging living cells and simultaneously quantifying the first viral binding events is difficult. Here, we show an atomic force and confocal microscopy set-up that allows the surface receptor landscape of cells to be imaged and the virus binding events within the first millisecond of contact with the cell to be mapped at high resolution (virus and cell surface receptors. We find that the first bond formed between the viral glycoprotein and its cognate cell surface receptor has relatively low lifetime and free energy, but this increases as additional bonds form rapidly (≤1 ms). The formation of additional bonds occurs with positive allosteric modulation and the three binding sites of the viral glycoprotein are quickly occupied. Our quantitative approach can be readily applied to study the binding of other viruses to animal cells.

  13. The ligand binding domain controls glucocorticoid receptor dynamics independent of ligand release.

    Science.gov (United States)

    Meijsing, Sebastiaan H; Elbi, Cem; Luecke, Hans F; Hager, Gordon L; Yamamoto, Keith R

    2007-04-01

    Ligand binding to the glucocorticoid receptor (GR) results in receptor binding to glucocorticoid response elements (GREs) and the formation of transcriptional regulatory complexes. Equally important, these complexes are continuously disassembled, with active processes driving GR off GREs. We found that co-chaperone p23-dependent disruption of GR-driven transcription depended on the ligand binding domain (LBD). Next, we examined the importance of the LBD and of ligand dissociation in GR-GRE dissociation in living cells. We showed in fluorescence recovery after photobleaching studies that dissociation of GR from GREs is faster in the absence of the LBD. Furthermore, GR interaction with a target promoter revealed ligand-specific exchange rates. However, using covalently binding ligands, we demonstrated that ligand dissociation is not required for receptor dissociation from GREs. Overall, these studies showed that activities impinging on the LBD regulate GR exchange with GREs but that the dissociation of GR from GREs is independent from ligand dissociation.

  14. Involvement of 1,25D{sub 3}-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Richard, Cynthia L. [Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Farach-Carson, Mary C.; Rohe, Ben [Department of Biological Sciences, University of Delaware, Newark, DE 19716 (United States); Nemere, Ilka [Department of Nutrition and Food Sciences, Center for Integrated BioSystems, Utah State University, Logan, UT 84322 8700 (United States); Meckling, Kelly A., E-mail: kmecklin@uoguelph.ca [Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada)

    2010-03-10

    In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D{sub 3} [1,25(OH){sub 2}D{sub 3}] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH){sub 2}D{sub 3} traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH){sub 2}D{sub 3} called 1,25D{sub 3}-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D{sub 3}-MARRS expression modulates 1,25(OH){sub 2}D{sub 3} activity in breast cancer cells. Relative levels of 1,25D{sub 3}-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D{sub 3}-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH){sub 2}D{sub 3} in MCF-7 cells, a ribozyme construct designed to knock down 1,25D{sub 3}-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D{sub 3}-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH){sub 2}D{sub 3} ( IC{sub 50} 56 {+-} 24 nM) compared to controls (319 {+-} 181 nM; P < 0.05). Reduction in 1,25D{sub 3}-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH){sub 2}D{sub 3}. Knockdown of 1,25D{sub 3}-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D{sub 3}-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH){sub 2}D{sub 3} in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D{sub 3}-MARRS expression or activity as anticancer agents.

  15. Expression and function of nicotinic acetylcholine receptors in stem cells

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  16. Binding properties of solubilized gonadotropin-releasing hormone receptor: role of carboxylic groups

    Energy Technology Data Exchange (ETDEWEB)

    Hazum, E.

    1987-11-03

    The interaction of /sup 125/I-buserelin, a superactive agonist of gonadotropin-releasing hormone (GnRH), with solubilized GnRH receptor was studied. The highest specific binding of /sup 125/I-buserelin to solubilized GnRH receptor is evident at 4/sup 0/C, and equilibrium is reached after 2 h of incubation. The soluble receptor retained 100% of the original binding activity when kept at 4 or 22/sup 0/C for 60 min. Mono- and divalent cations inhibited, in a concentration-dependent manner, the binding of /sup 125/I-buserelin to solubilized GnRH receptor. Monovalent cations require higher concentrations than divalent cations to inhibit the binding. Since the order of potency with the divalent cations was identical with that of their association constants to dicarboxylic compounds, it is suggested that there are at least two carboxylic groups of the receptor that participate in the binding of the hormone. The carboxyl groups of sialic acid residues are not absolutely required for GnRH binding since the binding of /sup 125/I-buserelin to solubilized GnRH receptor was only slightly affected by pretreatment with neuraminidase and wheat germ agglutinin. The finding that polylysines stimulate luteinizing hormone (LH) release from pituitary cell cultures with the same efficacy as GnRH suggest that simple charge interactions can induce LH release. According to these results, the authors propose that the driving force for the formation of the hormone-receptor complex is an ionic interaction between the positively charged amino acid arginine in position 8 and the carboxyl groups in the binding site.

  17. Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression.

    Science.gov (United States)

    Jones, Bart G; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Neale, Geoff; Gearhart, Patricia J; Hurwitz, Julia L

    2016-09-01

    Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease.

  18. Binding of white spot syndrome virus to Artemia sp. cell membranes.

    Science.gov (United States)

    Feng, Shuying; Li, Guangda; Feng, Wenpo; Huang, Jie

    2013-10-01

    Using differential velocity centrifugation, cell membranes of Artemia sp. were prepared, and their binding to white spot syndrome virus (WSSV) was analyzed in vitro. The results indicated that WSSV can specifically bind to Artemia cell membranes, and that WSSV receptor very likely existed in this membrane, which suggested that Artemia sp. may be a reservoir of WSSV. This study investigated the specific WSSV binding site by performing competitive inhibition experiments using shrimp gill cell membranes to bind WSSV to Artemia cell membranes. The results showed that shrimp gill cell membranes had a distinct inhibition effect on the specific binding of Artemia cell membranes to WSSV. Thus, potentially similar WSSV receptors or binding sites existed on Artemia sp. cell membranes and shrimp gill cell membranes. Taken together, these findings may provide experimental basis for the development of an effective approach to controlling WSSV, and theoretical basis for the study of WSSV receptors.

  19. Binding of GTPgamma[35S] is regulated by GDP and receptor activation. Studies with the nociceptin/orphanin FQ receptor.

    Science.gov (United States)

    McDonald, John; Lambert, David G

    2010-03-01

    We have examined the effects of ligand efficacy and receptor density on the binding of guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) and GDP to the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP)-coupled G-proteins. In GTPgamma[(35)S] binding experiments, using stable (CHO(hNOP)) and inducible (CHO(INDhNOP)) recombinant human and rat NOP we have measured: (i) ligand-specific GDP requirements; (ii) the effects of receptor density on guanine nucleotide affinity/capacity; and (iii) the effect of ligand efficacy on GTPgammaS association kinetics. GTPgammaS competition curves were shallow and modelled by high- and low-affinity components that were relatively consistent between cell types and tissue preparations. In the presence of 1 microM N/OFQ a high-affinity GDP binding site was also present, but the fraction of total binding was reduced. In an efficacy-dependent manner, the partial agonists [F/G]N/OFQ(1-13)NH(2) ([Phe(1)psi(CH(2)-NH)Gly(2)]-nociceptin(1-13)NH(2)) and naloxone benzoylhydrazone both reduced the fraction of high-affinity sites for GDP (relative to basal). While the pIC(50) for high-affinity GDP binding site did not decrease in the presence of 1 microM N/OFQ, N/OFQ produced a significant reduction in pIC(50) for the low-affinity site. Agonist-mediated decrease in affinity for GDP binding was efficacy-dependent. GDP displayed three affinities: high, conserved in the presence and absence of ligand; intermediate, present as a low fraction under basal conditions; low (efficacy-dependent), present during receptor activation representing the majority of binding. The affinity of GTPgamma[(35)S] was regulated by GDP and receptor activation caused increased binding of GTPgamma[(35)S] through a reduction in GDP affinity.

  20. A large-scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand-binding domains of taste receptor.

    Science.gov (United States)

    Yamashita, Atsuko; Nango, Eriko; Ashikawa, Yuji

    2017-08-19

    Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo- or heteromeric multimer protein complexes. Successful recombinant production of such proteins is often achieved by co-expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large-scale expression of hetero-multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high-expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large-scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes. © 2017 The Protein Society.

  1. Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: treprostinil is a potent DP1 and EP2 agonist.

    Science.gov (United States)

    Whittle, Brendan J; Silverstein, Adam M; Mottola, David M; Clapp, Lucie H

    2012-07-01

    The prostacyclin analogues, iloprost and treprostinil are extensively used in treating pulmonary hypertension. Their binding profile and corresponding biochemical cellular responses on human prostanoid receptors expressed in cell lines, have now been compared. Iloprost had high binding affinity for EP1 and IP receptors (Ki 1.1 and 3.9 nM, respectively), low affinity for FP, EP3 or EP4 receptors, and very low affinity for EP2, DP1 or TP receptors. By contrast, treprostinil had high affinity for the DP1, EP2 and IP receptors (Ki 4.4, 3.6 and 32 nM, respectively), low affinity for EP1 and EP4 receptors and even lower affinity for EP3, FP and TP receptors. In functional assays, iloprost had similar high activity in elevating cyclic AMP levels in cells expressing the human IP receptor and stimulating calcium influx in cells expressing EP1 receptors (EC50 0.37 and 0.3 nM, respectively) with the rank order of activity on the other receptors comparable to the binding assays. As with binding studies, treprostinil elevated cyclic AMP with a similar high potency in cells expressing DP1, IP and EP2 receptors (EC50 0.6, 1.9 and 6.2 nM, respectively), but had low activity at the other receptors. Activation of IP, DP1 and EP2 receptors, as with treprostinil, can all result in vasodilatation of human pulmonary arteries. However, activation of EP1 receptors can provoke vasoconstriction, and hence may offset the IP-receptor mediated vasodilator effects of iloprost. Treprostinil may therefore differ from iloprost in its overall beneficial pulmonary vasorelaxant profile and other pharmacological actions, especially in diseases where the IP receptor is down-regulated. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Collagen binding specificity of the discoidin domain receptors: Binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1

    OpenAIRE

    Xu, Huifang; Raynal, Nicolas; Stathopoulos, Stavros; Myllyharju, Johanna; Farndale, Richard W.; Leitinger, Birgit

    2011-01-01

    The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I–III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the...

  3. Upregulation of peroxisome proliferator-activated receptors and liver fatty acid binding protein in hepatic cells of broiler chicken supplemented with conjugated linoleic acids

    Directory of Open Access Journals (Sweden)

    Suriya Kumari Ramiah

    2015-09-01

    Full Text Available Since conjugated linoleic acid (CLA has structural and physiological characteristics similar to peroxisome proliferators, it is hypothesized that CLA would upregulate peroxisome proliferator-activated receptor (PPAR and liver fatty acid binding protein (LFABP in the liver of broiler chicken. The aim of the present study was to determine fatty acid composition of liver in CLA-fed broiler chickens and the genes associated with hepatic lipid metabolism. A total of 180-day-old broiler chicks were randomly assigned to two diets containing 0 and 2.5% CLA and fed for 6 weeks. Fatty acid (FA composition of liver and PPAR α and γ and L-FABP were analyzed. It has been demonstrated that CLA was found in the liver of CLA-feed chicken compared to control group. Hepatic PPAR α and γ mRNA levels were upregulated 1.2 and 3-fold in CLA-fed chickens compared to chickens fed diet without CLA respectively. A similar response of upregulation was observed for L-FABP mRNA expression. Our data highlights the role of PPARs as a core regulator in the regulation of lipid metabolism in chicken liver.

  4. Selectivity in progesterone and androgen receptor binding of progestagens used in oral contraceptives

    Energy Technology Data Exchange (ETDEWEB)

    Kloosterboer, H.J.; Vonk-Noordegraaf, C.A.; Turpijn, E.W.

    1988-09-01

    The relative binding affinities (RBAs) of four progestational compounds (norethisterone, levonorgestrel, 3-keto-desogestrel and gestodene) for the human progesterone and androgen receptors were measured in MCF-7 cytosol and intact MCF-7 cells. For the binding to the progesterone receptor, both Org 2058 and Org 3236 (or 3-keto-desogestrel) were used as labelled ligands. The following ranking (low to high) for the RBA of the nuclear (intact cells) progesterone receptor irrespective of the ligand used is found: norethisterone much less than levonorgestrel less than 3-keto-destogestrel less than gestodene. The difference between the various progestagens is significant with the exception of that between 3-keto-desogestrel and gestodene, when Org 2058 is used as ligand. For the cytosolic progesterone receptor, the same order is found with the exception that similar RBAs are found for gestodene and 3-keto-desogestrel. The four progestagens clearly differ with respect to binding to the androgen receptor using dihydrotestosterone as labelled ligand in intact cells; the ranking (low to high) is: norethisterone less than 3 keto-desogestrel less than levonorgestrel and gestodene. The difference between 3-keto-desogestrel and levonorgestrel or gestodene is significant. The selectivity indices (ratio of the mean RBA for the progesterone receptor to that of androgen receptor) in intact cells are significantly higher for 3-keto-desogestrel and gestodene than for levonorgestrel and norethisterone. From these results we conclude that the introduction of the 18-methyl in norethisterone (levonorgestel) increases both the binding to the progesterone and androgen receptors.

  5. Review of the Third Domain Receptor Binding Fragment of Alpha-fetoprotein (AFP): Plausible Binding of AFP to Lysophospholipid Receptor Targets.

    Science.gov (United States)

    Mizejewski, G J

    2016-01-31

    Alpha-fetoprotein (AFP) is a 69 kD fetal- and tumor-associated single-chain glycoprotein belonging to the albuminoid gene family. AFP functions as a carrier/transport molecule as well as a growth regulator and has been utilized as a clinical biomarker for both fetal defects and cancer growth. Lysophospholipids (LPLs) are plasma membrane-derived bioactive lipid signaling mediators composed of a small molecular weight single acyl carbon chain (palmitic, oleic acid) attached to a polar headgroup; they range in molecular mass from 250-750 daltons. The LPLs consist of either sphingosine-1-phosphate or lysophosphatidic acid, and mostly their choline, ethanolamine, serine or inositol derivatives. They are present only in vertebrates. These bioactive paracrine lipid mediators are ubiquitously distributed in tissues and are released from many different cell types (platelets, macrophages, monocytes, etc.) involved in developmental, physiological, and pathological processes. The LPLs bind to four different classes of G-protein coupled receptors described herein which transduce a multiple of cell effects encompassing activities such as morphogenesis, neural development, angiogenesis, and carcinogenesis. The identification of potential binding sites of LPL receptors on the AFP third domain receptor binding fragment were derived by computer modeling analysis. It is conceivable, but not proven, that AFP might bind not only to the LPL receptors, but also to LPLs themselves since AFP binds medium and long chain fatty acids. It is proposed that some of the activities ascribed to AFP in the past might be due in part to the presence of bound LPLs and/or their receptors.

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

    Energy Technology Data Exchange (ETDEWEB)

    Magno, Aaron L. [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Ingley, Evan [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Brown, Suzanne J. [Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Conigrave, Arthur D. [School of Molecular Bioscience, University of Sydney, New South Wales 2000 (Australia); Ratajczak, Thomas [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Ward, Bryan K., E-mail: bryanw@cyllene.uwa.edu.au [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia)

    2011-09-09

    Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} 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.

  7. Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-[alpha

    Energy Technology Data Exchange (ETDEWEB)

    Carter, Percy H.; Scherle, Peggy A.; Muckelbauer, Jodi K.; Voss, Matthew E.; Liu, Rui-qin; Thompson III, Lorin A.; Xu, Meizhong; Lo, Yvonne C.; Li, Zhong; Strzemienski, Paul; Yang, Gengjie; Falahatpishen, Nikoo; Farrow, Neil A.; Tebben, Andrew J.; Underwood, Denis; Trzaskos, James M.; Friedman, Steven M.; Newton, Robert C.; Decicco, Carl P. (DuPont)

    2010-03-05

    The binding of tumor necrosis factor alpha (TNF-{alpha}) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-{alpha} to TNFRc1 (IC{sub 50} = 50 nM) and also blocked TNF-stimulated phosphorylation of I{kappa}-B in Ramos cells (IC{sub 50} = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 {micro}M. Detailed evaluation of this and related molecules revealed that compounds in this class are 'photochemically enhanced' inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 mM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-{alpha} to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-{alpha}-TNFRc1 interaction.

  8. Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-alpha.

    Science.gov (United States)

    Carter, P H; Scherle, P A; Muckelbauer, J K; Voss, M E; Liu, R Q; Thompson, L A; Tebben, A J; Solomon, K A; Lo, Y C; Li, Z; Strzemienski, P; Yang, G; Falahatpisheh, N; Xu, M; Wu, Z; Farrow, N A; Ramnarayan, K; Wang, J; Rideout, D; Yalamoori, V; Domaille, P; Underwood, D J; Trzaskos, J M; Friedman, S M; Newton, R C; Decicco, C P; Muckelbauer, J A

    2001-10-09

    The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.

  9. Structure and Mode of Peptide Binding of Pheromone Receptor PrgZ

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Schuurman-Wolters, Gea K.; Dunny, Gary; Slotboom, Dirk-Jan; Poolman, Bert

    2012-01-01

    Wepresent the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and Opp

  10. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2011-01-01

    Full Text Available Abstract Background The surface glycoprotein (SU, gp120 of the human immunodeficiency virus (HIV must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP to bind the Duffy Antigen Receptor for Chemokines (DARC and invade reticulocytes. Results Variable loop 3 (V3 of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket.

  11. Binding of the influenza A virus to cell-surface receptors: structures of five hemagglutinin-sialyloligosaccharide complexes determined by X-ray crystallography.

    Science.gov (United States)

    Eisen, M B; Sabesan, S; Skehel, J J; Wiley, D C

    1997-05-26

    The structures of five complexes of the X-31 influenza A (H3N2) virus hemagglutinin with sialyloligosaccharide receptor analogs have been determined from 2.5 to 2.8 A resolution by X-ray crystallography. There is well-defined electron density for three to five saccharides in all five complexes and a striking conformational difference between two linear pentasaccharides with the same composition but different linkage [alpha(2-->6) or alpha(2-->3)] at the terminal sialic acid. The bound position of the terminal sialic acid (NeuAc) is the same in all five complexes and is identical to that reported previously from the study of mono- and trisaccharides. The two oligosaccharides with NeuAc alpha(2-->6)Gal linkages and GlcNAc at the third position have a folded conformation with the GlcNAc doubled back to contact the sialic acid. The pentasaccharide with a terminal NeuAc alpha(2-->3)Gal linkage and GlcNAc at the third position has an extended (not folded) conformation and exits from the opposite side of the binding site than the alpha(2-->6)-linked molecule of the same composition. The difference between the conformation of the pentasaccharide with a 2,6 linkage and the trisaccharide 2,6-sialyllactose suggests that 2,6-sialyllactose is not, as previously believed, an appropriate analog of natural influenza A virus receptors. The oligosaccharides studied are NeuAc alpha(2-->3)Gal beta(1-->4)Glc, NeuAc alpha(2-->6)Gal beta(1-->4)Glc, NeuAc alpha(2-->3)Gal beta(1-->3)GlcNAc beta(1-->3)Gal beta(1-->4)Glc, NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Gal beta(1-->4)Glc, and [NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc]2 beta(1-->3/6)Gal-beta-O-(CH2)5-COOCH3.

  12. Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca2+-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus

    Science.gov (United States)

    Rivera, Patricia; Arrabal, Sergio; Cifuentes, Manuel; Grondona, Jesús M.; Pérez-Martín, Margarita; Rubio, Leticia; Vargas, Antonio; Serrano, Antonia; Pavón, Francisco J.; Suárez, Juan; Rodríguez de Fonseca, Fernando

    2014-01-01

    The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca2+ and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca2+-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB+1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin+ cells (granular and pyramidal neurons), and calretinin+ and parvalbumin+ interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin+ principal cells in the dentate gyrus and CA1, and in the calretinin+ and parvalbumin+ interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL+ terminals were only observed around CA1 calbindin+ pyramidal cells, CA1/3 calretinin+ interneurons and CA3 parvalbumin+ interneurons localized in the pyramidal cell layers. Interestingly, calbindin+ pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions. PMID:25018703

  13. Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

    Science.gov (United States)

    Rivera, Patricia; Arrabal, Sergio; Cifuentes, Manuel; Grondona, Jesús M; Pérez-Martín, Margarita; Rubio, Leticia; Vargas, Antonio; Serrano, Antonia; Pavón, Francisco J; Suárez, Juan; Rodríguez de Fonseca, Fernando

    2014-01-01

    The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca(2+) and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca(2+)-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB(+) 1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin(+) cells (granular and pyramidal neurons), and calretinin(+) and parvalbumin(+) interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin(+) principal cells in the dentate gyrus and CA1, and in the calretinin(+) and parvalbumin(+) interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL(+) terminals were only observed around CA1 calbindin(+) pyramidal cells, CA1/3 calretinin(+) interneurons and CA3 parvalbumin(+) interneurons localized in the pyramidal cell layers. Interestingly, calbindin(+) pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.

  14. Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα and degrading (MAGL, FAAH enzymes in cells expressing the Ca2+-binding proteins calbindin, calretinin and parvalbumin in the adult rat hippocampus

    Directory of Open Access Journals (Sweden)

    Patricia eRivera

    2014-06-01

    Full Text Available The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca2+ and the activation of specific 2-AG synthesizing (i.e. DAGLα enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca2+-binding proteins (CaBPs is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL and FAAH and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. CB1, DAGLα and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB1+ fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin+ cells (granular and pyramidal neurons, and calretinin+ and parvalbumin+ interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin+ principal cells in the dentate gyrus and CA1, and in the calretinin+ and parvalbumin+ interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL+ terminals were only observed around CA1 calbindin+ pyramidal cells, CA1/3 calretinin+ interneurons and CA3 parvalbumin+ interneurons localized in the pyramidal cell layers. Interestingly, calbindin+ pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.

  15. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  16. Changes of insulin effect on lipogenesis and insulin binding receptors during hypokinesia

    Science.gov (United States)

    Macho, L.; Fickova, M.; Zorad, S.

    The effect of hypokinesia on insulin action and insulin binding to specific receptors in fat cells was studied. Male Wistar rats were exposed to hypokinesia in special adjustable plastic cages for 1, 7, 21 and 60 days, and the stimulatory effect of insulin (10 and 100 mU) on the incorporation of radiocarbon labelled glucose into lipids of fat tissue and the binding of insulin to receptors of isolated adipocytes was estimated. The stimulation of lipogenesis by insulin was slightly diminished after hypokinesia for 1 day, however, an important increase of insulin action was found in rats exposed to hypokinesia for 60 days. The decrease of insulin binding capacity of the number of binding sites per cell and of the insulin receptor density was found after 1 day of hypokinesia. In rats exposed to hypokinesia for 60 days, in agreement with the higher stimulatory affect of insulin, an increase of insulin receptor density was observed. These results showed that hypokinesia has an important influence on stimulatory action of insulin and on insulin receptors in adipocytes.

  17. Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca2+-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus

    Science.gov (United States)

    Rivera, Patricia; Arrabal, Sergio; Vargas, Antonio; Blanco, Eduardo; Serrano, Antonia; Pavón, Francisco J.; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2014-01-01

    The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca2+ fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca2+-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα+/calbindin+ cells were closely surrounded by NAPE-PLD+ fiber varicosities. No pyramidal PPARα+/calbindin+ cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD+/calretinin+ cells were specifically detected in CA3. NAPE-PLD+ puncta surrounded the calretinin+ cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions. PMID:24672435

  18. Insulin-like growth factor II: complexity of biosynthesis and receptor binding

    DEFF Research Database (Denmark)

    Gammeltoft, S; Christiansen, Jan; Nielsen, F C

    1991-01-01

    , Man-6-P induces cellular responses. We have studied rat brain neuronal precursor cells where Man-6-P acted as a mitogen suggesting that phosphomannosylated proteins may act as growth factors via the Man-6-P/IGF-II receptor. In conclusion, the gene expression and mechanism of action of IGF-II is very...... and the mannose-6-phosphate (Man-6-P)/IGF-II receptor. There is consensus that the cellular effects of IGF-II are mediated by the IGF-I receptor via activation of its intrinsic tyrosine kinase. The Man-6-P/IGF-II receptor is involved in endocytosis of lysosomal enzymes and IGF-II. In selected cell types, however...... complex suggesting that its biological actions can be regulated at different levels including the transcription, translation, posttranslational processing, receptor binding and intracellular signalling....

  19. Computational Characterization and Prediction of Estrogen Receptor Coactivator Binding Site Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Bennion, B J; Kulp, K S; Cosman, M; Lightstone, F C

    2005-08-26

    Many carcinogens have been shown to cause tissue specific tumors in animal models. The mechanism for this specificity has not been fully elucidated and is usually attributed to differences in organ metabolism. For heterocyclic amines, potent carcinogens that are formed in well-done meat, the ability to either bind to the estrogen receptor and activate or inhibit an estrogenic response will have a major impact on carcinogenicity. Here we describe our work with the human estrogen receptor alpha (hERa) and the mutagenic/carcinogenic heterocyclic amines PhIP, MeIQx, IFP, and the hydroxylated metabolite of PhIP, N2-hydroxy-PhIP. We found that PhIP, in contrast to the other heterocyclic amines, increased cell-proliferation in MCF-7 human breast cancer cells and activated the hERa receptor. We show mechanistic data supporting this activation both computationally by homology modeling and docking, and by NMR confirmation that PhIP binds with the ligand binding domain (LBD). This binding competes with estradiol (E2) in the native E2 binding cavity of the receptor. We also find that other heterocyclic amines and N2-hydroxy-PhIP inhibit ER activation presumably by binding into another cavity on the LBD. Moreover, molecular dynamics simulations of inhibitory heterocyclic amines reveal a disruption of the surface of the receptor protein involved with protein-protein signaling. We therefore propose that the mechanism for the tissue specific carcinogenicity seen in the rat breast tumors and the presumptive human breast cancer associated with the consumption of well-done meat maybe mediated by this receptor activation.

  20. Single chain human interleukin 5 and its asymmetric mutagenesis for mapping receptor binding sites.

    Science.gov (United States)

    Li, J; Cook, R; Dede, K; Chaiken, I

    1996-01-26

    Wild type human (h) interleukin 5 (wt IL5) is composed of two identical peptide chains linked by disulfide bonds. A gene encoding a single chain form of hIL5 dimer was constructed by linking the two hIL5 chain coding regions with Gly-Gly linker. Expression of this gene in COS cells yielded a single chain IL5 protein (sc IL5) having biological activity similar to that of wt IL5, as judged by stimulation of human cell proliferation. Single chain and wt IL5 also had similar binding affinity for soluble IL5 receptor alpha chain, the specificity subunit of the IL5 receptor, as measured kinetically with an optical biosensor. The design of functionally active sc IL5 molecule. Such mutagenesis was exemplified by changes at residues Glu-13, Arg-91, Glu-110, and Trp-111. The receptor binding and bioactivity data obtained are consistent with a model in which residues from both IL5 monomers interact with the receptor alpha chain, while the interaction likely is asymmetric due to the intrinsic asymmetry of folded receptor. The results demonstrate a general route to the further mapping of receptor and other binding sites on the surface of human IL5.

  1. Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry

    Institute of Scientific and Technical Information of China (English)

    Jizhen Wang; Balaji Manicassamy; Michael Caffrey; Lijun Rong

    2011-01-01

    Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality.Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells,followed by fusion of virus-cell membrane also mediated by GP.Using an human immunodeficiency virus (HIV)-based pseudotyping system,the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions.We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry.An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry.It was found that R64 and K95 are involved in receptor binding.In contrast,some residues such as I170 are important for viral entry,but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data,suggesting that these residues are involved in post-binding steps of viral entry.Furthermore,our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

  2. Structure-activity relations in binding of perfluoroalkyl compounds to human thyroid hormone T3 receptor.

    Science.gov (United States)

    Ren, Xiao-Min; Zhang, Yin-Feng; Guo, Liang-Hong; Qin, Zhan-Fen; Lv, Qi-Yan; Zhang, Lian-Ying

    2015-02-01

    Perfluoroalkyl compounds (PFCs) have been shown to disrupt thyroid functions through thyroid hormone receptor (TR)-mediated pathways, but direct binding of PFCs with TR has not been demonstrated. We investigated the binding interactions of 16 structurally diverse PFCs with human TR, their activities on TR in cells, and the activity of perfluorooctane sulfonate (PFOS) in vivo. In fluorescence competitive binding assays, most of the 16 PFCs were found to bind to TR with relative binding potency in the range of 0.0003-0.05 compared with triiodothyronine (T3). A structure-binding relationship for PFCs was observed, where fluorinated alkyl chain length longer than ten, and an acid end group were optimal for TR binding. In thyroid hormone (TH)-responsive cell proliferation assays, PFOS, perfluorohexadecanoic acid, and perfluorooctadecanoic acid exhibited agonistic activity by promoting cell growth. Furthermore, similar to T3, PFOS exposure promoted expression of three TH upregulated genes and inhibited three TH downregulated genes in amphibians. Molecular docking analysis revealed that most of the tested PFCs efficiently fit into the T3-binding pocket in TR and formed a hydrogen bond with arginine 228 in a manner similar to T3. The combined in vitro, in vivo, and computational data strongly suggest that some PFCs disrupt the normal activity of TR pathways by directly binding to TR.

  3. Neurotoxin Binding Site on the Acetylcholine Receptor

    Science.gov (United States)

    1987-03-01

    Neurotoxins fron snakes belonging to the families Elapidae (cobras, kraits, mambas , and others) and Hydrophidae (sea snakes) are polypep- tides of 60-74... black mice were used as immunizing anrmals and as a soirce of feeder spleen cells. Mice were injected subcutaneously with j2-mer (50 nmol) emulsified in

  4. Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lianying [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); College of Life Science, Dezhou University, Dezhou 253023 (China); Ren, Xiao-Min; Wan, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China)

    2014-09-15

    Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.

  5. Specific binding of benzodiazepines to human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    1999-01-01

    Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.

  6. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, Ole Nørregaard; Engelholm, L H

    2000-01-01

    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

  7. Quercetin enhances apoptotic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in ovarian cancer cells through reactive oxygen species (ROS) mediated CCAAT enhancer-binding protein homologous protein (CHOP)-death receptor 5 pathway

    Science.gov (United States)

    Yi, Liu; Zongyuan, Yang; Cheng, Gong; Lingyun, Zhang; GuiLian, Yu; Wei, Gong

    2014-01-01

    Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown efficacy in a phase 2 clinical trial, development of resistance to TRAIL by tumor cells is a major roadblock. We investigated whether quercetin, a flavonoid, can sensitize human ovarian cancer cells to TRAIL. Results indicate that quercetin sensitized cancer cells to TRAIL. The quercetin induced expression of death receptor DR5 but did not affect expression of DR4 in cancer cells. The induction of DR5 was mediated through activation of JNK and through upregulation of a transcription factor CCAAT enhancer-binding protein homologous protein (CHOP); as silencing of these signaling molecules abrogated the effect of quercetin. Upregulation of DR5 was mediated through the generation of reactive oxygen species (ROS), as ROS scavengers reduced the effect of quercetin on JNK activation, CHOP upregulation, DR induction, TRAIL sensitization, downregulated the expression of cell survival proteins and upregulated the proapoptotic proteins. Furthermore, quercetin enhances TRAIL mediated inhibition of tumor growth of human SKOV-3 xenograft was associated with induction of apoptosis, activation of caspase-3, CHOP and DR5. Overall, our data suggest that quercetin enhances apoptotic death of ovarian cancer cells to TRAIL through upregulation of CHOP-induced DR5 expression following ROS mediated endoplasmic reticulum-stress. PMID:24612139

  8. New insights into pb5, the receptor binding protein of bacteriophage T5, and its interaction with its Escherichia coli receptor FhuA

    OpenAIRE

    Flayhan, Ali; Wien, Frank; Paternostre, Maïté; Boulanger, Pascale; Breyton, Cécile

    2012-01-01

    International audience; The majority of bacterial viruses are bacteriophages bearing a tail that serves to recognise the bacterial surface and deliver the genome into the host cell. Infection is initiated by the irreversible interaction between the viral receptor binding protein (RBP) and a receptor at the surface of the bacterium. This interaction results ultimately in the phage DNA release in the host cytoplasm. Phage T5 infects Escher-ichia coli after binding of its RBP pb5 to the outer me...

  9. Agonist Binding to Chemosensory Receptors: A Systematic Bioinformatics Analysis

    Directory of Open Access Journals (Sweden)

    Fabrizio Fierro

    2017-09-01

    Full Text Available Human G-protein coupled receptors (hGPCRs constitute a large and highly pharmaceutically relevant membrane receptor superfamily. About half of the hGPCRs' family members are chemosensory receptors, involved in bitter taste and olfaction, along with a variety of other physiological processes. Hence these receptors constitute promising targets for pharmaceutical intervention. Molecular modeling has been so far the most important tool to get insights on agonist binding and receptor activation. Here we investigate both aspects by bioinformatics-based predictions across all bitter taste and odorant receptors for which site-directed mutagenesis data are available. First, we observe that state-of-the-art homology modeling combined with previously used docking procedures turned out to reproduce only a limited fraction of ligand/receptor interactions inferred by experiments. This is most probably caused by the low sequence identity with available structural templates, which limits the accuracy of the protein model and in particular of the side-chains' orientations. Methods which transcend the limited sampling of the conformational space of docking may improve the predictions. As an example corroborating this, we review here multi-scale simulations from our lab and show that, for the three complexes studied so far, they significantly enhance the predictive power of the computational approach. Second, our bioinformatics analysis provides support to previous claims that several residues, including those at positions 1.50, 2.50, and 7.52, are involved in receptor activation.

  10. Distinct ETA receptor binding mode of macitentan as determined by site directed mutagenesis.

    Directory of Open Access Journals (Sweden)

    John Gatfield

    Full Text Available The competitive endothelin receptor antagonists (ERA bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min occupancy half-lives at the ET(A receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ET(A receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ET(A receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ET(A receptor-antagonist interaction modes, we performed functional studies using ET(A receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ET(A receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that--in contrast to bosentan and ambrisentan--macitentan-ET(A receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan's sustained target occupancy and

  11. Crosstalk between the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and the vitamin D receptor (VDR) in human breast cancer cells: PPAR{gamma} binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} mediated transactivation

    Energy Technology Data Exchange (ETDEWEB)

    Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang; Mehta, Rajeshwari R. [Cancer Biology Division, IIT Research Institute, 10 West 35th Street, Chicago, IL 60616 (United States); Knethen, Andreas von [Institute of Biochemistry, Johann Wolfgang Goethe University, Frankfurt (Germany); Choubey, Divaker [Department of Environmental Health, University of Cincinnati, 3223 Eden Avenue, P.O. Box 670056, Cincinnati, OH 45267 (United States); Mehta, Rajendra G., E-mail: rmehta@iitri.org [Cancer Biology Division, IIT Research Institute, 10 West 35th Street, Chicago, IL 60616 (United States)

    2012-11-15

    Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ER{alpha}) physically binds to peroxisome proliferator-activated receptor gamma (PPAR{gamma}) and inhibits its transcriptional activity. The interaction between PPAR{gamma} and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPAR{gamma} and VDR signaling, and for the first time we show that PPAR{gamma} physically associates with VDR in human breast cancer cells. We found that overexpression of PPAR{gamma} decreased 1{alpha},25-dihydroxyvitamin D{sub 3} (1,25D{sub 3}) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPAR{gamma}'s hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPAR{gamma}'s AF2 domain attenuated its repressive action on 1,25D{sub 3} transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPAR{gamma} was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXR{alpha}). Overexpression of RXR{alpha} blocked PPAR{gamma}'s suppressive effect on 1,25D{sub 3} action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPAR{gamma} and VDR pathways. -- Highlights: PPAR{gamma}'s role on 1{alpha},25-dihydroxyvitamin D{sub 3} transcriptional activity is examined. Black-Right-Pointing-Pointer PPAR{gamma} physically binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} action. Black-Right-Pointing-Pointer PPAR{gamma}'s hinge and ligand binding domains are important for this inhibitory effect. Black-Right-Pointing-Pointer PPAR{gamma} competes with VDR for the availability of their binding

  12. Receptor-binding domain of ephrin-A1: production in bacterial expression system and activity.

    Science.gov (United States)

    Nekrasova, O V; Sharonov, G V; Tikhonov, R V; Kolosov, P M; Astapova, M V; Yakimov, S A; Tagvey, A I; Korchagina, A A; Bocharova, O V; Wulfson, A N; Feofanov, A V; Kirpichnikov, M P

    2012-12-01

    Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.

  13. Marlin-1, a novel RNA-binding protein associates with GABA receptors.

    Science.gov (United States)

    Couve, Andrés; Restituito, Sophie; Brandon, Julia M; Charles, Kelly J; Bawagan, Hinayana; Freeman, Katie B; Pangalos, Menelas N; Calver, Andrew R; Moss, Stephen J

    2004-04-02

    GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission.

  14. De novo analysis of receptor binding affinity data of xanthine adenosine receptor antagonists.

    Science.gov (United States)

    Dalpiaz, A; Gardenghi, A; Borea, P A

    1995-03-01

    The receptor binding affinity data to adenosine A1 and A2 receptors of a wide series of xanthine derivatives have been analyzed by means of the Free-Wilson model. The analysis of the individual group contribution shows, for both A1 and A2 receptors, the primary importance of the presence of bulky substituents at position 8 for an optimum receptor binding. Moreover, considering the different aij contributions of bulky substituents at position 8 for affinity to A1 with respect to A2 receptors, this position appears to be the most important for the synthesis of highly A1 selective xanthine derivatives. Moreover the analysis of group contributions for other substitution positions of the xanthine moiety allows to state that suitable substitutions at positions 3 and 7 could confer some degree of A2 selectivity.

  15. Glycophorin B is the erythrocyte receptor of Plasmodium falciparum erythrocyte-binding ligand, EBL-1.

    Science.gov (United States)

    Mayer, D C Ghislaine; Cofie, Joann; Jiang, Lubin; Hartl, Daniel L; Tracy, Erin; Kabat, Juraj; Mendoza, Laurence H; Miller, Louis H

    2009-03-31

    In the war against Plasmodium, humans have evolved to eliminate or modify proteins on the erythrocyte surface that serve as receptors for parasite invasion, such as the Duffy blood group, a receptor for Plasmodium vivax, and the Gerbich-negative modification of glycophorin C for Plasmodium falciparum. In turn, the parasite counters with expansion and diversification of ligand families. The high degree of polymorphism in glycophorin B found in malaria-endemic regions suggests that it also may be a receptor for Plasmodium, but, to date, none has been identified. We provide evidence from erythrocyte-binding that glycophorin B is a receptor for the P. falciparum protein EBL-1, a member of the Duffy-binding-like erythrocyte-binding protein (DBL-EBP) receptor family. The erythrocyte-binding domain, region 2 of EBL-1, expressed on CHO-K1 cells, bound glycophorin B(+) but not glycophorin B-null erythrocytes. In addition, glycophorin B(+) but not glycophorin B-null erythrocytes adsorbed native EBL-1 from the P. falciparum culture supernatants. Interestingly, the Efe pygmies of the Ituri forest in the Democratic Republic of the Congo have the highest gene frequency of glycophorin B-null in the world, raising the possibility that the DBL-EBP family may have expanded in response to the high frequency of glycophorin B-null in the population.

  16. Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

    Indian Academy of Sciences (India)

    Pidaran Murugan; Leelavinothan Pari; Chippada Appa Rao

    2008-03-01

    Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)–nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin–receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (Kd1), low affinity (Kd2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is

  17. Cell-surface acceleration of urokinase-catalyzed receptor cleavage

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Ploug, M; Behrendt, N

    1997-01-01

    The urokinase-type plasminogen activator (uPA) binds to a specific cell-surface receptor, uPAR. On several cell types uPAR is present both in the full-length form and a cleaved form, uPAR(2+3), which is devoid of binding activity. The formation of uPAR(2+3) on cultured U937 cells is either direct...

  18. [3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors.

    Science.gov (United States)

    Yagle, Monica A; Martin, Michael W; de Fiebre, Christopher M; de Fiebre, NancyEllen C; Drewe, John A; Dillon, Glenn H

    2003-12-01

    Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.

  19. Receptor binding specificity of recent human H3N2 influenza viruses

    Directory of Open Access Journals (Sweden)

    Cummings Richard D

    2007-05-01

    Full Text Available Abstract Background Human influenza viruses are known to bind to sialic acid linked α2-6 to galactose, but the binding specificity beyond that linkage has not been systematically examined. H3N2 human influenza isolates lost binding to chicken red cells in the 1990s but viruses isolated since 2003 have re-acquired the ability to agglutinate chicken erythrocytes. We have investigated specificity of binding, changes in hemagglutinin sequence of the recent viruses and the role of sialic acid in productive infection. Results Viruses that agglutinate, or do not agglutinate, chicken red cells show identical binding to a Glycan Array of 264 oligosaccharides, binding exclusively to a subset of α2-6-sialylsaccharides. We identified an amino acid change in hemagglutinin that seemed to correlate with chicken red cell binding but when tested by mutagenesis there was no effect. Recombinant hemagglutinins expressed on Sf-9 cells bound chicken red cells but the released recombinant baculoviruses agglutinated only human red cells. Similarly, an isolate that does not agglutinate chicken red cells show hemadsorption of chicken red cells to infected MDCK cells. We suggest that binding of chicken red cells to cell surface hemagglutinin but not to virions is due to a more favorable hemagglutinin density on the cell surface. We investigated whether a virus specific for α2-6 sialyloligosaccharides shows differential entry into cells that have varying proportions of α2-6 and α2-3 sialic acids, including human A549 and HeLa cells with high levels of α2-6 sialic acid, and CHO cells that have only α2-3 sialic acid. We found that the virus enters all cell types tested and synthesizes viral nucleoprotein, localized in the nucleus, and hemagglutinin, transported to the cell surface, but infectious progeny viruses were released only from MDCK cells. Conclusion Agglutination of chicken red cells does not correlate with altered binding to any oligosaccharide on the Glycan

  20. Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.

    Science.gov (United States)

    Castro, Marián; Nikolaev, Viacheslav O; Palm, Dieter; Lohse, Martin J; Vilardaga, Jean-Pierre

    2005-11-01

    Parathyroid hormone (PTH) and its related receptor (PTHR) are essential regulators of calcium homeostasis and bone physiology. PTH activates PTHR by interacting with a ligand-binding site localized within the N-terminal extracellular domain (the N-domain) and the domain comprising the seven transmembrane helices and the connecting extracellular loops (the J-domain). PTH binding triggers a conformational switch in the receptor, leading to receptor activation and subsequent cellular responses. The process of receptor activation occurs rapidly, within approximately 1 s, but the binding event preceding receptor activation is not understood. By recording FRET between tetramethyl-rhodamine in PTH(1-34) and GFP in the N-domain of the receptor, we measured the binding event in real time in living cells. We show that the association time course between PTH(1-34) and PTHR involves a two-step binding process where the agonist initially binds the receptor with a fast time constant (tau approximately 140 ms) and then with slower kinetics (tau approximately 1 s). The fast and slow phases were assigned to hormone association to the receptor N- and J domains, respectively. Our data indicate that the slow binding step to the J-domain coincides with a conformational switch in the receptor, also monitored by FRET between the enhanced cyan fluorescent protein and the enhanced yellow fluorescent protein in the PTHR sensor, PTHR enhanced cyan fluorescent protein/enhanced yellow fluorescent protein (PTHR(CFP/YFP)). These data suggest that the conformational change that switches the receptor into its active state proceeds in a sequential manner, with the first rapid binding step event preceding receptor activation by PTH(1-34).

  1. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

    Science.gov (United States)

    Freund, Jacquelyn; May, Rebecca M; Yang, Enjun; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K; Kambayashi, Taku

    2016-08-01

    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

  2. Soy isoflavones increase quinone reductase in hepa-1c1c7 cells via estrogen receptor beta and nuclear factor erythroid 2-related factor 2 binding to the antioxidant response element.

    Science.gov (United States)

    Froyen, Erik B; Steinberg, Francene M

    2011-09-01

    Soy protein and isoflavones (genistein and daidzein) have been demonstrated to increase quinone reductase (QR) activity, protein, and mRNA in animal and cell culture models. However, their mechanism of action has not been completely characterized. Additionally, it has not been determined if equol, a daidzein metabolite, can modulate QR activity and expression. Estrogen receptor beta (ERβ) is thought to be involved in stimulating QR gene transcription by anti-estrogens and phytoestrogens, along with nuclear factor erythroid 2-related factor 2 (Nrf2). This study tested the hypothesis that genistein, daidzein and equol increase quinone reductase activity, protein and mRNA via ERβ and Nrf2 binding to the QR antioxidant response element (ARE). QR expression and activity were determined using TaqMan polymerase chain reaction, protein immunoblots and activity assays. Molecular events were investigated using luciferase reporter gene assays and chromatin immunoprecipitation (ChIP). Hepa-1c1c7 cells were treated with control [0.1% (v:v) dimethyl sulfoxide (DMSO)]; 1 μmol/L β-naphthoflavone (positive control); 5 μmol/L resveratrol (ChIP positive control for ERβ binding) and 1, 5 and 25 μmol/L genistein, daidzein or equol. Treatment durations were 1 h (ChIP), 24 h (mRNA and luciferase assays) and 24 and 48 h (protein and activity). Genistein, daidzein and equol increased QR activity, protein and mRNA, with daidzein and equol having more of an impact at physiologic concentrations (1 and 5 μmol/L) compared to genistein. Furthermore, the study results demonstrate that genistein, daidzein and equol interact with the QR ARE and that daidzein and equol act via both ERβ and Nrf2 binding strongly to the QR ARE.

  3. The predicted 3D structure of the human D2 dopamine receptor and the binding site and binding affinities for agonists and antagonists

    Science.gov (United States)

    Kalani, M. Yashar S.; Vaidehi, Nagarajan; Hall, Spencer E.; Trabanino, Rene J.; Freddolino, Peter L.; Kalani, Maziyar A.; Floriano, Wely B.; Tak Kam, Victor Wai; Goddard, William A., III

    2004-03-01

    Dopamine neurotransmitter and its receptors play a critical role in the cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's disease and schizophrenia would be significantly enhanced with the availability of the 3D structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. We report here the 3D structure of the long isoform of the human D2 dopamine receptor, predicted from primary sequence using first-principles theoretical and computational techniques (i.e., we did not use bioinformatic or experimental 3D structural information in predicting structures). The predicted 3D structure is validated by comparison of the predicted binding site and the relative binding affinities of dopamine, three known dopamine agonists (antiparkinsonian), and seven known antagonists (antipsychotic) in the D2 receptor to experimentally determined values. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies, and give relative binding affinities that correlate well with experiments. The predicted binding site for dopamine and agonists is located between transmembrane (TM) helices 3, 4, 5, and 6, whereas the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM helix 5. We identify characteristic differences between the binding sites of agonists and antagonists.

  4. Development and application of a nonradioactive binding assay of oxidized low-density lipoprotein to macrophage scavenger receptors

    Science.gov (United States)

    Montano, Erica N.; Boullier, Agnès; Almazan, Felicidad; Binder, Christoph J.; Witztum, Joseph L.; Hartvigsen, Karsten

    2013-01-01

    Macrophages play a key role in atherogenesis in part through excessive uptake of oxidized LDL (OxLDL) via scavenger receptors. Binding of OxLDL to macrophages has traditionally been assessed using radiolabeled OxLDL. To allow more efficient and convenient measurements, we developed a nonradioactive binding assay in which biotinylated OxLDL (Bt-OxLDL) is added to macrophages in 96-well microtiter culture plates under various conditions and the extent of binding is determined using solid phase chemiluminescent immunoassay techniques. As examples, we show that Bt-OxLDL displayed high and saturable binding to macrophages in contrast to Bt-LDL, which showed very low binding. In competition assays, unlabeled OxLDL and the anti-OxLDL monoclonal antibody E06 inhibited Bt-OxLDL binding to macrophages in a dose-dependent manner. Specific binding of Bt-OxLDL to ApoE/SR-A/CD36 triple knockout macrophages was reduced by 80% as compared with binding to macrophages from ApoE knockout mice. Binding of Bt-OxLDL to CD36 transfected COS-7 cells showed enhanced saturable binding compared with mock-transfected cells. This assay avoids the use of radioactivity and uses small amounts of materials. It can be used to study binding of OxLDL to macrophages and factors that influence this binding. The techniques described should be readily adaptable to study of other ligands, receptors, and cell types. PMID:23997238

  5. Ovarian cancer cell heparan sulfate 6-O-sulfotransferases regulate an angiogenic program induced by heparin-binding epidermal growth factor (EGF)-like growth factor/EGF receptor signaling.

    Science.gov (United States)

    Cole, Claire L; Rushton, Graham; Jayson, Gordon C; Avizienyte, Egle

    2014-04-11

    Heparan sulfate (HS) is a component of cell surface and extracellular matrix proteoglycans that regulates numerous signaling pathways by binding and activating multiple growth factors and chemokines. The amount and pattern of HS sulfation are key determinants for the assembly of the trimolecular, HS-growth factor-receptor, signaling complex. Here we demonstrate that HS 6-O-sulfotransferases 1 and 2 (HS6ST-1 and HS6ST-2), which perform sulfation at 6-O position in glucosamine in HS, impact ovarian cancer angiogenesis through the HS-dependent HB-EGF/EGFR axis that subsequently modulates the expression of multiple angiogenic cytokines. Down-regulation of HS6ST-1 or HS6ST-2 in human ovarian cancer cell lines results in 30-50% reduction in glucosamine 6-O-sulfate levels in HS, impairing HB-EGF-dependent EGFR signaling and diminishing FGF2, IL-6, and IL-8 mRNA and protein levels in cancer cells. These cancer cell-related changes reduce endothelial cell signaling and tubule formation in vitro. In vivo, the development of subcutaneous tumor nodules with reduced 6-O-sulfation is significantly delayed at the initial stages of tumor establishment with further reduction in angiogenesis occurring throughout tumor growth. Our results show that in addition to the critical role that 6-O-sulfate moieties play in angiogenic cytokine activation, HS 6-O-sulfation level, determined by the expression of HS6ST isoforms in ovarian cancer cells, is a major regulator of angiogenic program in ovarian cancer cells impacting HB-EGF signaling and subsequent expression of angiogenic cytokines by cancer cells.

  6. Both host and parasite MIF molecules bind to chicken macrophages via CD74 surface receptor.

    Science.gov (United States)

    Kim, Sungwon; Cox, Chasity M; Jenkins, Mark C; Fetterer, Ray H; Miska, Katarzyna B; Dalloul, Rami A

    2014-12-01

    Macrophage migration inhibitory factor (MIF) is recognized as a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. Our group has identified both chicken and Eimeria MIFs, and characterized their function in enhancing innate immune responses during inflammation. In this study, we report that chicken CD74 (ChCD74), a type II transmembrane protein, functions as a macrophage surface receptor that binds to MIF molecules. First, to examine the binding of MIF to chicken monocytes/macrophages, fresh isolated chicken peripheral blood mononuclear cells (PBMCs) were stimulated with rChIFN-γ and then incubated with recombinant chicken MIF (rChMIF). Immunofluorescence staining with anti-ChMIF followed by flow cytometry revealed the binding of MIF to stimulated PBMCs. To verify that ChCD74 acts as a surface receptor for MIF molecules, full-length ChCD74p41 was cloned, expressed and its recombinant protein (rChCD74p41) transiently over-expressed with green fluorescent protein in chicken fibroblast DF-1 cells. Fluorescence analysis revealed a higher population of cells double positive for CD74p41 and rChMIF, indicating the binding of rChMIF to DF-1 cells via rChCD74p41. Using a similar approach, it was found that Eimeria MIF (EMIF), which is secreted by Eimeria sp. during infection, bound to chicken macrophages via ChCD74p41 as a surface receptor. Together, this study provides conclusive evidence that both host and parasite MIF molecules bind to chicken macrophages via the surface receptor ChCD74.

  7. Differential expression of functional Fc-receptors and additional immune complex receptors on mouse kidney cells.

    Science.gov (United States)

    Suwanichkul, Adisak; Wenderfer, Scott E

    2013-12-01

    The precise mechanisms by which circulating immune complexes accumulate in the kidney to form deposits in glomerulonephritis are not well understood. In particular, the role of resident cells within glomeruli of the kidney has been widely debated. Immune complexes have been shown to bind one glomerular cell type (mesangial cells) leading to functional responses such as pro-inflammatory cytokine production. To further assess the presence of functional immunoreceptors on resident glomerular cells, cultured mouse renal epithelial, endothelial, and mesangial cells were treated with heat-aggregated mouse IgG or preformed murine immune complexes. Mesangial and renal endothelial cells were found to bind IgG complexes, whereas glomerular epithelial cell binding was minimal. A blocking antibody for Fc-gamma receptors reduced binding to mesangial cells but not renal endothelial cells, suggesting differential immunoreceptor utilization. RT-PCR and immunostaining based screening of cultured renal endothelial cells showed limited low-level expression of known Fc-receptors and Ig binding proteins. The interaction between mesangial cells and renal endothelial cells and immune complexes resulted in distinct, cell-specific patterns of chemokine and cytokine production. This novel pathway involving renal endothelial cells likely contributes to the predilection of circulating immune complex accumulation within the kidney and to the inflammatory responses that drive kidney injury.

  8. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

    Energy Technology Data Exchange (ETDEWEB)

    Strauch, Eva-Maria; Bernard, Steffen M.; La, David; Bohn, Alan J.; Lee, Peter S.; Anderson, Caitlin E.; Nieusma, Travis; Holstein, Carly A.; Garcia, Natalie K.; Hooper, Kathryn A.; Ravichandran, Rashmi; Nelson, Jorgen W.; Sheffler, William; Bloom, Jesse D.; Lee, Kelly K.; Ward, Andrew B.; Yager, Paul; Fuller, Deborah H.; Wilson, Ian A.; Baker , David (UWASH); (Scripps); (FHCRC)

    2017-06-12

    Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

  9. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

    Science.gov (United States)

    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( BSC cultures treated with 10 n TBA exhibit increased ( BSC cultures.

  10. Oxidative stress effect on progesterone-induced blocking factor (PIBF) binding to PIBF-receptor in lymphocytes.

    Science.gov (United States)

    de la Haba, Carlos; Palacio, José R; Palkovics, Tamas; Szekeres-Barthó, Júlia; Morros, Antoni; Martínez, Paz

    2014-01-01

    Receptor-ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor-ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces lipid raft clustering.

  11. On the use of nonfluorescent dye labeled ligands in FRET-based receptor binding studies.

    Science.gov (United States)

    Tahtaoui, Chouaib; Guillier, Fabrice; Klotz, Philippe; Galzi, Jean-Luc; Hibert, Marcel; Ilien, Brigitte

    2005-12-01

    The efficiency of fluorescence resonance energy transfer (FRET) is dependent upon donor-acceptor proximity and spectral overlap, whether the acceptor partner is fluorescent or not. We report here on the design, synthesis, and characterization of two novel pirenzepine derivatives that were coupled to patent blue VF and pinacyanol dyes. These nonfluorescent compounds, when added to cells stably expressing enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors, promote EGFP fluorescence extinction in a time-, concentration-, and atropine-dependent manner. They display nanomolar affinity for the muscarinic receptor, determined using either FRET or classical radioligand binding conditions. We provide evidence that these compounds behave as potent acceptors of energy from excited EGFP with quenching efficiencies comparable to those of analogous fluorescent bodipy or rhodamine red pirenzepine derivatives. The advantages they offer over fluorescent ligands are illustrated and discussed in terms of reliability, sensitivity, and wider applicability of FRET-based receptor binding assays.

  12. FcRn expression, ligands binding properties and its regulation in human immune cells and hepatocytes

    OpenAIRE

    2007-01-01

    ABSTRACT Expression and diverse functions of MHC class I related neonatal Fc receptor in different tissues is continually reported. To contribute to the understanding of how the receptor functions according to cell type, we investigated the expression and ligands binding properties of FcRn in human immune cells and hepatocytes. Here, we report that heterodimeric FcRn is expressed in these cells as evidenced by RT-PCR, Western immunoblottting and flow cytometry. The receptor expression i...

  13. The Binding Ability Analysis of the Normal VLDL Receptor and Its Mutant

    Institute of Scientific and Technical Information of China (English)

    QU Shen; FENG Ning; LIU Zhiguo; ZHOU Hua; DENG Yaozu; FENG Zongchen

    2001-01-01

    The ligand-binding domain of VLDL receptor contains eight imperfectly similar repeats.To discuss the contribution of each repeat to ligand binding, the RT-PCR technique was used to clone the VLDLR-cDNA from the heart muscle of Chinese people. Two recombinants were further constructed, which contained the full-length cDNA of VLDLR and the mutant lacking repeats 1-5.CHO cell line was transfected with two recombinants. The expression of VLDLR gene could be detected by RT-PCR from the CHO cells transfected with pCD-VR. The results of binding experiments showed that the ability of the CHO cells transfected with the full-length cDNA of VLDL-R binding DiI-labeled β-VLDL was higher than that of the CHO cells transfected with the mutant. Our findings indicated that human VLDL-R gene could be expressed effectively on CHO cells, and the receptor was almost inactivated when repeats1-5 were deleted.

  14. Fluorescence Resonance Energy Transfer Imaging Reveals that Chemokine-Binding Modulates Heterodimers of CXCR4 and CCR5 Receptors

    OpenAIRE

    2008-01-01

    BACKGROUND: Dimerization has emerged as an important feature of chemokine G-protein-coupled receptors. CXCR4 and CCR5 regulate leukocyte chemotaxis and also serve as a co-receptor for HIV entry. Both receptors are recruited to the immunological synapse during T-cell activation. However, it is not clear whether they form heterodimers and whether ligand binding modulates the dimer formation. METHODOLOGY/PRINCIPAL FINDINGS: Using a sensitive Fluorescence Resonance Energy Transfer (FRET) imaging ...

  15. Cell cycle phase regulates glucocorticoid receptor function.

    Directory of Open Access Journals (Sweden)

    Laura Matthews

    Full Text Available The glucocorticoid receptor (GR is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. In contrast to many other nuclear receptors, GR is thought to be exclusively cytoplasmic in quiescent cells, and only translocate to the nucleus on ligand binding. We now demonstrate significant nuclear GR in the absence of ligand, which requires nuclear localisation signal 1 (NLS1. Live cell imaging reveals dramatic GR import into the nucleus through interphase and rapid exclusion of the GR from the nucleus at the onset of mitosis, which persists into early G(1. This suggests that the heterogeneity in GR distribution is reflective of cell cycle phase. The impact of cell cycle-driven GR trafficking on a panel of glucocorticoid actions was profiled. In G2/M-enriched cells there was marked prolongation of glucocorticoid-induced ERK activation. This was accompanied by DNA template-specific, ligand-independent GR transactivation. Using chimeric and domain-deleted receptors we demonstrate that this transactivation effect is mediated by the AF1 transactivation domain. AF-1 harbours multiple phosphorylation sites, which are consensus sequences for kinases including CDKs, whose activity changes during the cell cycle. In G2/M there was clear ligand independent induction of GR phosphorylation on residues 203 and 211, both of which are phosphorylated after ligand activation. Ligand-independent transactivation required induction of phospho-S211GR but not S203GR, thereby directly linking cell cycle driven GR modification with altered GR function. Cell cycle phase therefore regulates GR localisation and post-translational modification which selectively impacts GR activity. This suggests that cell cycle phase is an important determinant in the cellular response to Gc, and that mitotic index contributes to tissue Gc sensitivity.

  16. The intact urokinase receptor is required for efficient vitronectin binding

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Behrendt, N; Ploug, M;

    1997-01-01

    The urokinase receptor (uPAR) is a receptor for both urokinase plasminogen activator (uPA) and the adhesion protein vitronectin. There are two forms of cell surface-bound uPAR; intact uPAR and a cleaved form, uPAR(2+3), which is formed by uPA-catalyzed cleavage of uPAR. In ligand-blotting experim...

  17. Binding of concanavalin A by the cell membrane of a unicellular organism, Paramecium aurelia.

    Science.gov (United States)

    Wyroba, E

    1975-01-01

    As demonstrated by electron microscopy, Concanavalin A receptors exist on the cell membrane of Paramecium aurelia. The interpretation of the cytochemical detection of Con A binding is not very precise because of a slight, unspecific peroxidase adsorption.

  18. Binding thermodynamics at the human cannabinoid CB1 and CB2 receptors.

    Science.gov (United States)

    Merighi, Stefania; Simioni, Carolina; Gessi, Stefania; Varani, Katia; Borea, Pier Andrea

    2010-02-01

    The thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees of the binding equilibrium of agonists and antagonists at cannabinoid CB(1) and CB(2) receptors were determined by means of affinity measurements at different temperatures and van't Hoff plots were constructed. Affinity constants were measured on CHO cells transfected with the human CB(1) and CB(2) receptors by inhibition assays of the binding of the cannabinoid receptor agonist [(3)H]-CP-55,940. van't Hoff plots were linear for agonists and antagonists in the temperature range 0-30 degrees C. The thermodynamic parameters for CB(1) receptors fall in the ranges 17< or =DeltaH degrees < or =59 kJ/mol and 213< or =DeltaS degrees < or =361 kJ/mol for agonists and -52< or =DeltaH degrees < or =-26 kJ/mol and -12< or =DeltaS degrees < or =38 kJ/mol for antagonists. The thermodynamic parameters for CB(2) receptors fall in the ranges 27< or =DeltaH degrees < or =48 kJ/mol and 234< or =DeltaS degrees < or =300 kJ/mol for agonists and -19< or =DeltaH degrees < or =-17 kJ/mol and 43< or =DeltaS degrees < or =74 kJ/mol for antagonists. Collectively, these data show that agonist binding is always totally entropy-driven while antagonist binding is enthalpy and entropy-driven, indicating that CB(1) and CB(2) receptors are thermodynamically discriminated. These data could give new details on the nature of the forces driving the CB(1) and CB(2) binding at a molecular level. Enthalpy, entropy, free energy and binding affinity for each ligand to its receptor can all be assessed and therefore the optimal binding profile discovered. Carrying out these binding investigations as early as possible in the discovery process increases the probability that a lead compound will become a successful pharmaceutical compound.

  19. Insulin-Like Growth Factor Binding Protein 2 (IGFBP-2) Promotes Growth and Survival of Breast Epithelial Cells: Novel Regulation of the Estrogen Receptor

    National Research Council Canada - National Science Library

    Foulstone, Emily J; Zeng, Li; Perks, Claire M; Holly, Jeff M. P

    2013-01-01

    .... However, how IGFBP-2 contributes to the malignant state is still unclear. Silencing IGFBP-2 blocked cell proliferation and in MCF-7 cells increased cell death, indicating that IGFBP-2 was acting in both a mitogenic and a survival capacity...

  20. Binding of levomepromazine and cyamemazine to human recombinant dopamine receptor subtypes

    Directory of Open Access Journals (Sweden)

    Lalit K. Srivastava

    Full Text Available Background and Objectives: Clozapine (CLOZ and levomepromazine (LMP improve treatment-resistant schizophrenia. The superior efficacy of CLOZ compared with other antipsychotic agents has been attributed to an effect on D1-like and D4 receptors. We examined the binding of LMP, CLOZ and cyamemazine (CMZ, a neuroleptic analog of LMP, to human recombinant dopamine (rDA receptor subtypes. Methods: Binding studies were performed on frozen membrane suspensions of human rDA receptor subtypes expressed in Sf9 cells. Results: (i LMP has a high affinity (Ki, nM for rD2 receptor subtypes (rD2L 8.6; rD2S 4.3; rD3 8.3; rD4.2 7.9; (ii LMP and CLOZ have comparable affinities for the rD1 receptor (54.3 vs 34.6; (iii CMZ has high affinities for rD2-like and rD1-like receptors (rD2L 4.6; rD2S 3.3; rD3 6.2; rD4.2 8.5; rD1 3.9; rD5 10.7; (iv CMZ is 9 times more potent than CLOZ at the rD1 receptor and 5 times more potent than CLOZ at the rD4.2 receptor; (v CMZ has high affinities for rD1 and rD5 receptor subtypes compared with LMP and CLOZ. Conclusions: If D1 and D4 receptors are important sites for the unique action of CLOZ, the present study points to a need for clinical trials comparing CMZ with CLOZ in schizophrenia and in particular, treatment-resistant schizophrenia, especially given the risk for agranulocytosis with CLOZ.

  1. Binding of levomepromazine and cyamemazine to human recombinant dopamine receptor subtypes

    Directory of Open Access Journals (Sweden)

    Lalit K. Srivastava

    2009-09-01

    Full Text Available Background and Objectives: Clozapine (CLOZ and levomepromazine (LMP improve treatment-resistant schizophrenia. The superior efficacy of CLOZ compared with other antipsychotic agents has been attributed to an effect on D1-like and D4 receptors. We examined the binding of LMP, CLOZ and cyamemazine (CMZ, a neuroleptic analog of LMP, to human recombinant dopamine (rDA receptor subtypes. Methods: Binding studies were performed on frozen membrane suspensions of human rDA receptor subtypes expressed in Sf9 cells. Results: (i LMP has a high affinity (Ki, nM for rD2 receptor subtypes (rD2L 8.6; rD2S 4.3; rD3 8.3; rD4.2 7.9; (ii LMP and CLOZ have comparable affinities for the rD1 receptor (54.3 vs 34.6; (iii CMZ has high affinities for rD2-like and rD1-like receptors (rD2L 4.6; rD2S 3.3; rD3 6.2; rD4.2 8.5; rD1 3.9; rD5 10.7; (iv CMZ is 9 times more potent than CLOZ at the rD1 receptor and 5 times more potent than CLOZ at the rD4.2 receptor; (v CMZ has high affinities for rD1 and rD5 receptor subtypes compared with LMP and CLOZ. Conclusions: If D1 and D4 receptors are important sites for the unique action of CLOZ, the present study points to a need for clinical trials comparing CMZ with CLOZ in schizophrenia and in particular, treatment-resistant schizophrenia, especially given the risk for agranulocytosis with CLOZ.

  2. Effect of phospholipid hydrolysis by phospholipase A2 on the kinetics of antagonist binding to cardiac muscarinic receptors.

    Science.gov (United States)

    Rauch, B; Niroomand, F; Messineo, F C; Weis, A; Kübler, W; Hasselbach, W

    1994-09-15

    Activation of phospholipases during prolonged myocardial ischemia could contribute to the functional derangement of myocardial cells by altering the phospholipid environment of a number of membrane bound proteins including receptors. The present study examined the kinetics of muscarinic receptor antagonist [3H]quinuclidinyl benzilate binding ([3H]QNB) to muscarinic receptors of highly purified sarcolemmal membranes under control conditions and after treatment with phospholipase A2 (PLA2; EC 3.1.1.4). Initial binding rates of QNB exhibited saturation kinetics, when plotted against the ligand concentration in control and PLA2 treated sarcolemmal membranes. This kinetic behaviour of QNB-binding is consistent with at least a two step binding mechanism. According to this two step binding hypothesis an unstable intermediate receptor-QNB complex (R*QNB) forms rapidly, and this form undergoes a slow conversion to the high affinity ligand-receptor complex R-QNB. The Michaelis constant Km of R-QNB formation was 1.8 nM, whereas the dissociation constant Kd obtained from equilibrium measurements was 0.062 nM. After 5 min exposure of sarcolemmal membranes to PLA2QNB binding capacity (Bmax) was reduced by 62%, and the affinity of the remaining receptor sites was decreased by 47% (Kd = 0.116 nM). This PLA2-induced increase of Kd was accompanied by a corresponding increase of Km, whereas the rate constants k2 and k-2 of the hypothetical slow conversion step (second reaction step) remained unchanged. These results suggest that binding of QNB to cardiac muscarinic receptors induces a transition in the receptor-ligand configuration, which is necessary for the formation of the final high affinity R-QNB complex. PLA2-induced changes of the lipid environment result in the inability of a part of the receptor population to undergo this transition, thereby inhibiting high affinity QNB-binding.

  3. The stereoisomers of 17{alpha}-[{sup 123}I]iodovinyloestradiol and its 11{beta}-methoxy derivative evaluated for their oestrogen receptor binding in human MCF-7 cells and rat uterus, and their distribution in immature rats

    Energy Technology Data Exchange (ETDEWEB)

    Rijks, L.J.M. [Department of Nuclear Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam (Netherlands); Boer, G.J. [Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam (Netherlands); Endert, E. [Department of Endocrinology, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam (Netherlands); Bruin, K. de [Department of Nuclear Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam (Netherlands); Bos, J.C. van den [Cygne BV and Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Doremalen, P.A.P.M. van [Cygne BV and Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Schoonen, W.G.E.J. [Department of Endocrinology, NV Organon, P.O. Box 20, 5340 BH Oss (Netherlands); Janssen, A.G.M. [Cygne BV and Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Royen, E.A. van [Department of Nuclear Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam (Netherlands)

    1996-03-01

    We studied the potential of both stereoisomers of 17{alpha}-[{sup 123}I]iodovinyloestradiol (E- and Z-[{sup 123}I]IVE) and of 11{beta}-methoxy-17{alpha}-[{sup 123}I]iodovinyloestradiol (E- and Z-[{sup 123}I]MIVE) as suitable radioligands for the imaging of oestrogen receptor(ER)-positive human breast tumours. The 17{alpha}-[{sup 123}I]iodovinyloestradiols were prepared stereospecifically by oxidative radio-iododestannylation of the corresponding 17{alpha}-tri-n-butylstannylvinyloestradiol precursors. Competitive binding studies were performed in order to determine the relative binding affinity (RBA) of the unlabelled 17{alpha}-iodovinyloestradiols for the ER in both human MCF-7 breast tumour cells and rat uterine tissue, compared with that of diethylstilboestrol (DES). Target tissue uptake, retention and uptake selectivity of their {sup 123}I-labelled analogues were studied in immature female rats. All four 17{alpha}-iodovinyloestradiols showed high affinity for the ER in human MCF-7 cells, as well as rat uterus. Their RBA for the ER showed the following order of decreasing potency: RBA of DES >Z-IVE >Z-MIVE >E-MIVE {>=}E-IVE. Neither of these 17{alpha}-iodovinyloestradiols showed any significant binding to the sex hormone binding globulin in human plasma. The biodistribution studies showed ER-mediated uptake in the uterus, ovaries and pituitary, that of E- and Z-[{sup 123}I]MIVE being higher than that of E- and Z-[{sup 123}I]IVE. High target-to-non-target tissue uptake ratios, especially at longer periods after injection (up to 24 h), were exhibited by both isomers of [{sup 123}I]MIVE. The uterus-to-blood uptake ratio was higher for E-[{sup 123}I]MIVE. However, the uterus-to-fat uptake ratio appeared to be higher for the Z-isomer of [{sup 123}I]MIVE, especially at 24 h after injection. (orig./MG). With 5 figs., 5 tabs.

  4. Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM receptors. Overall, the N-terminus of chemokine receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two major chemokine subgroups occur, as CC-chemokines mainly......The human chemokine system comprises 19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis...... and surveillance. Chemokines are a group of 8-12 kDa large peptides with a secondary structure consisting of a flexible N-terminus and a core-domain usually stabilized by two conserved disulfide bridges. They mainly interact with the extracellular domains of their cognate 7TM receptors. Affinityand activity...

  5. Receptor binding peptides for target-selective delivery of nanoparticles encapsulated drugs

    Directory of Open Access Journals (Sweden)

    Accardo A

    2014-03-01

    Full Text Available Antonella Accardo,1 Luigi Aloj,2 Michela Aurilio,2 Giancarlo Morelli,1 Diego Tesauro11Centro interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB, Department of Pharmacy and Istituto di Biostrutture e Bioimmagini - Consiglio Nazionale delle Ricerche (IBB CNR, University of Naples “Federico II”, 2Department of Nuclear Medicine, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione “G. Pascale”, Napoli, ItalyAbstract: Active targeting by means of drug encapsulated nanoparticles decorated with targeting bioactive moieties represents the next frontier in drug delivery; it reduces drug side effects and increases the therapeutic index. Peptides, based on their chemical and biological properties, could have a prevalent role to direct drug encapsulated nanoparticles, such as liposomes, micelles, or hard nanoparticles, toward the tumor tissues. A considerable number of molecular targets for peptides are either exclusively expressed or overexpressed on both cancer vasculature and cancer cells. They can be classified into three wide categories: integrins; growth factor receptors (GFRs; and G-protein coupled receptors (GPCRs. Therapeutic agents based on nanovectors decorated with peptides targeting membrane receptors belonging to the GPCR family overexpressed by cancer cells are reviewed in this article. The most studied targeting membrane receptors are considered: somatostatin receptors; cholecystokinin receptors; receptors associated with the Bombesin like peptides family; luteinizing hormone-releasing hormone receptors; and neurotensin receptors. Nanovectors of different sizes and shapes (micelles, liposomes, or hard nanoparticles loaded with doxorubicin or other cytotoxic drugs and externally functionalized with natural or synthetic peptides are able to target the overexpressed receptors and are described based on their formulation and in vitro and in vivo behaviors.Keywords: receptors binding peptides, drug delivery

  6. Angiotensin II Inhibits Insulin Binding to Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Su-Jin Oh

    2011-06-01

    Full Text Available BackgroundInsulin-mediated glucose uptake in insulin target tissues is correlated with interstitial insulin concentration, rather than plasma insulin concentration. Therefore, insulin delivery to the interstitium of target tissues is very important, and the endothelium may also play an important role in the development of insulin resistance.MethodsAfter treating bovine aortic endothelial cells with angiotensin II (ATII, we observed the changes in insulin binding capacity and the amounts of insulin receptor (IR on the cell membranes and in the cytosol.ResultsAfter treatment of 10-7M ATII, insulin binding was decreased progressively, up to 60% at 60 minutes (P<0.05. ATII receptor blocker (eprosartan dose dependently improved the insulin binding capacity which was reduced by ATII (P<0.05. At 200 µM, eprosartan fully restored insulin binding capacity, althogh it resulted in only a 20% to 30% restoration at the therapeutic concentration. ATII did not affect the total amount of IR, but it did reduce the amount of IR on the plasma membrane and increased that in the cytosol.ConclusionATII decreased the insulin binding capacity of the tested cells. ATII did not affect the total amount of IR but did decrease the amount of IR on the plasma membrane. Our data indicate that ATII decreases insulin binding by translocating IR from the plasma membrane to the cytosol. The binding of insulin to IR is important for insulin-induced vasodilation and transendothelial insulin transport. Therefore, ATII may cause insulin resistance through this endothelium-based mechanism.

  7. Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

    Energy Technology Data Exchange (ETDEWEB)

    Waser, Beatrice; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, PO Box 62, Berne (Switzerland)

    2014-06-15

    Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the {sup 125}iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer {sup 125}I-GLP-1(7-36)amide. Receptor autoradiography studies with {sup 125}I-GLP-1(7-36)amide agonist or {sup 125}I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist {sup 125}I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer {sup 125}I-GLP-1(7-36)amide. For comparison, {sup 125}I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with {sup 125}I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

  8. Study on Synthesis and Binding Ability of a New Anion Receptor Containing NH Binding Sites

    Institute of Scientific and Technical Information of China (English)

    QIAO,Yan-Hong; LIN,Hai; LIN,Hua-Kuan

    2007-01-01

    A new colorimetric recognition receptor 1 based on the dual capability containing NH binding sites of selectively sensing anionic guest species has been synthesized. Compared with other halide anions, its UV/Vis absorption spectrum in dimethyl sulfoxide showed the response toward the presence of fluoride anion with high selectivity,and also displayed dramatic color changes from colorless to yellow in the presence of TBAF (5 × 10-5 mol/L). The similar UV/Vis absorption spectrum change also occurred when 1 was treated with AcO- while a little change with H2PO-4 and OH-. Receptor 1 has almost not affinity abilities to Cl-, Br- and I-. The binding ability of receptor 1to fluoride with high selectivity over other halides contributes to the anion size and the ability of forming hydrogen bonding. While the different ability of binding with geometrically triangular (AcO-), tetrahedral (H2PO-4 ) and linear (OH-) anions maybe result from their geometry configuration.

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

    Science.gov (United States)

    Matrosovich, Mikhail; Klenk, Hans-Dieter

    2003-01-01

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

  10. Localization of CGRP receptor components and receptor binding sites in rhesus monkey brainstem

    DEFF Research Database (Denmark)

    Eftekhari, Sajedeh; Roberts, Rhonda; Chen, Tsing-Bau

    2016-01-01

    that several regions in the brainstem may be involved in CGRP signaling. Interestingly, we found receptor expression and antagonist binding in some areas that are not protected by the blood-brain barrier, which suggests that drugs inhibiting CGRP signaling may not be able to penetrate the central nervous...

  11. Structure of the homodimeric androgen receptor ligand-binding domain

    Science.gov (United States)

    Nadal, Marta; Prekovic, Stefan; Gallastegui, Nerea; Helsen, Christine; Abella, Montserrat; Zielinska, Karolina; Gay, Marina; Vilaseca, Marta; Taulès, Marta; Houtsmuller, Adriaan B.; van Royen, Martin E.; Claessens, Frank; Fuentes-Prior, Pablo; Estébanez-Perpiñá, Eva

    2017-01-01

    The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor. PMID:28165461

  12. Binding of lurasidone, a novel antipsychotic, to rat 5-HT7 receptor: analysis by [3H]SB-269970 autoradiography.

    Science.gov (United States)

    Horisawa, Tomoko; Ishiyama, Takeo; Ono, Michiko; Ishibashi, Tadashi; Taiji, Mutsuo

    2013-01-10

    Lurasidone is a novel antipsychotic agent with high affinity for dopamine D(2) and serotonin 5-HT(7), 5-HT(2A), and 5-HT(1A) receptors. We previously reported that in addition to its antipsychotic action, lurasidone shows beneficial effects on mood and cognition in rats, likely through 5-HT(7) receptor antagonistic actions. In this study, we evaluated binding of lurasidone to 5-HT(7) receptors in the rat brain by autoradiography using [(3)H]SB-269970, a specific radioligand for 5-HT(7) receptors. Brain slices were incubated with 4 nM [(3)H]SB-269970 at room temperature and exposed to imaging plates for 8 weeks before phosphorimager analysis. Using this method, we first investigated 5-HT(7) receptor distribution. We found that 5-HT(7) receptors are abundantly localized in brain limbic structures, including the lateral septum, thalamus, hypothalamus, hippocampus, and amygdala. On the other hand, its distribution was moderate in the cortex and low in the caudate putamen and cerebellum. Secondly, binding of lurasidone, a selective 5-HT(7) receptor antagonist SB-656104-A and an atypical antipsychotic olanzapine to this receptor was examined. Lurasidone, SB-656104-A (10–1000 nM), and olanzapine (100–10,000 nM) showed concentration-dependent inhibition of [(3)H]SB-269970 binding with IC(50) values of 90, 49, and 5200 nM, respectively. Similar inhibitory actions of these drugs were shown in in vitro [(3)H]SB-269970 binding to 5-HT(7) receptors expressed in Chinese hamster ovary cells. Since there was no marked species difference in rat and human 5-HT(7) receptor binding by lurasidone (K(i) = 1.55 and 2.10 nM, respectively), these findings suggest that binding to 5-HT(7) receptors might play some role in its beneficial pharmacological actions in schizophrenic patients.

  13. The Nucleotide-Binding Oligomerization Domain-Like Receptor Family Pyrin Domain-Containing 3 Inflammasome Regulates Bronchial Epithelial Cell Injury and Proapoptosis after Exposure to Biomass Fuel Smoke.

    Science.gov (United States)

    Li, Chen; Zhihong, Huang; Wenlong, Li; Xiaoyan, Liu; Qing, Chen; Wenzhi, Luo; Siming, Xie; Shengming, Liu

    2016-12-01

    The number of individuals in the population exposed to biomass fuel smoke (BS) is far greater than the number of cigarette smokers. About 20% of cigarette smokers develop chronic obstructive pulmonary disease (COPD) due to smoke-induced irreversible damage and sustained inflammation of the airway epithelium. However, the role of BS in COPD pathogenesis remains to be elucidated. In this study, we investigated the expression of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing (NLRP) 3 and caspase-1 in the bronchial epithelium from patients with COPD, and further determined the specific role of the NLRP3 inflammasome in bronchial epithelium injury using two in vitro models (BS and cigarette smoke [CS]) in the human bronchial epithelial (HBE) cell line (16HBE). After exposure to BS and CS, the release of damage-associated molecular patterns, the transcriptional and translational up-regulation of NLRP3, and the activation of caspase-1 were observed in cells at different time points. Because IL-1β secretion was dependent on the NLRP3 inflammasome, we assessed CXCL-8 production in response to smoke. Using a transwell migration assay in which 16HBE cells and human alveolar macrophages were cocultured, we showed that smoke-induced NLRP3 activation in 16HBE cells increased the migration of human alveolar macrophages. When the NLRP3 expression was silenced, the average migration distance of 16HBE was increased in scratch assay, because the activation of NLRP3 induced apoptosis by the p53-Bax mitochondrial pathway in the smoke-induced response. These results demonstrate the importance of the NLRP3 inflammasome in mediating BS- and CS-induced HBE cell damage and proapoptosis.

  14. The effect of neurotoxin on rabies virus binding to mouse neuroblastoma cells.

    Science.gov (United States)

    Briggs, D J; Phillips, R M

    1991-08-01

    Mouse neuroblastoma cells were exposed to alpha bungarotoxin, a neurotoxin known to inhibit rabies virus binding to the nicotinic acetylcholine receptor located at the neuromuscular junction in muscle tissue. The total amount of 3H-CVS virus that bound to neurotoxin treated cells was separated into specific and non-specific binding using a cold competition assay. Comparison of untreated and neurotoxin treated cells demonstrated that the majority of cell-associated virus in untreated cells was of a specific nature whereas the majority of the cell-associated virus in neurotoxin treated cells was due to non-specific binding.

  15. The binding of pirenzepine to digitonin-solubilized muscarinic acetylcholine receptors from the rat myocardium.

    Science.gov (United States)

    Birdsall, N. J.; Hulme, E. C.; Keen, M.

    1986-01-01

    The binding of pirenzepine to digitonin-solubilized rat myocardial muscarinic acetylcholine receptors has been examined at 4 degrees C. Solubilization produced only small changes in the binding of N-methylscopolamine and atropine. In contrast to the low affinity binding of pirenzepine found to be present in in the membranes, high affinity binding was detected in the soluble preparation. In both preparations, pirenzepine binding was complex. High affinity pirenzepine binding (KD approximately 3 X 10(-8)M) to the soluble myocardial receptors could be monitored directly using [3H]-pirenzepine. [3H]-pirenzepine-labelled soluble myocardial receptors have a sedimentation coefficient of 11.1 s. This indicates that [3H]-pirenzepine binds predominantly to the uncoupled form of the receptor. However, [3H]-pirenzepine-agonist competition experiments indicated that the high affinity pirenzepine binding sites are capable of coupling with a guanosine 5'-triphosphate (GTP)-binding protein. Pirenzepine affinities for the soluble myocardial receptors were unaffected by their state of association with the GTP-binding proteins found in the heart. The equilibrium binding properties of the soluble cortical and myocardial receptors were very similar. However, the binding kinetics of the myocardial receptor were much slower. It appears that the membrane environment can affect the affinity of pirenzepine for the rat myocardial muscarinic receptor. Removal of the constraint by solubilization allows the expression of high affinity pirenzepine binding. PMID:3754173

  16. Foreign or Domestic CARs: Receptor Ligands as Antigen-Binding Domains

    Directory of Open Access Journals (Sweden)

    Donald R. Shaffer

    2014-01-01

    Full Text Available Chimeric antigen receptors (CARs are increasingly being used in clinical trials to treat a variety of malignant conditions and recent results with CD19-specific CARs showing complete tumor regressions has sparked the interest of researchers and the public alike. Traditional CARs have been generated using single-chain variable fragments (scFv, often derived from murine monoclonal antibodies, for antigen specificity. As the clinical experience with CAR T cells grows, so does the potential for unwanted immune responses against the foreign transgene. Strategies that may reduce the immunogenicity of CAR T cells are humanization of the scFv and the use of naturally occurring receptor ligands as antigen-binding domains. Herein, we review the experience with alternatively designed CARs that contain receptor ligands rather than scFv. While most of the experiences have been in the pre-clinical setting, clinical data is also emerging.

  17. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, Ole Nørregaard; Engelholm, L H

    2000-01-01

    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition......The plasminogen activation cascade system, directed by urokinase and the urokinase receptor, plays a key role in extracellular proteolysis during tissue remodeling. To identify molecular interaction partners of these trigger proteins on the cell, we combined covalent protein cross-linking with mass...... spectrometry based methods for peptide mapping and primary structure analysis of electrophoretically isolated protein conjugates. A specific tri-molecular complex was observed upon addition of pro-urokinase to human U937 cells. This complex included the urokinase receptor, pro-urokinase, and an unknown, high...

  18. Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding

    DEFF Research Database (Denmark)

    Sap, J; Jiang, Y P; Friedlander, D

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.......-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable...... cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does...

  19. Analysis of Ligand Binding ErbB Receptor Tyrosine Kinases

    Science.gov (United States)

    1999-07-01

    Abraham , J.A., Miller, J., Fiddes, J.C. & Klagsbrun, M. (1991) A heparin- binding growth factor secreted by macrophage-like cells that is related...to EGF. Science 251, 936-939. 12 Anual Report 1999 DAMD17-98-1-8228 Principal Investigator; Ferguson, Kathryn, M. 22. Peles , E. & Yarden, Y. (1993) Neu

  20. Evidence for dual receptor-binding sites in Clostridium difficile toxin A.

    Science.gov (United States)

    Lambert, Gregory S; Baldwin, Michael R

    2016-12-01

    TcdA (308 kDa) and TcdB (270 kDa) disrupt the integrity of the intestinal epithelial barrier and provide an environment favorable for Clostridium difficile colonization. Recent evidence suggests that entry of TcdA into cells is mediated by at least two domains. Here, we report the characterization of a second receptor-binding domain (RBD2) for TcdA. While both the isolated combined repetitive oligopeptides (CROPs) and RBD2 fragments are rapidly internalized into cells under physiologic conditions, only the CROPs domain appreciably accumulates at the cell surface. Once internalized, CROPs and RBD2 are trafficked to late endosomal compartments. An internal deletion of RBD2 from TcdA holotoxin ablated toxicity in HT29 cells. These data are consistent with the recently proposed dual receptor model of cellular entry. © 2016 Federation of European Biochemical Societies.

  1. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    Energy Technology Data Exchange (ETDEWEB)

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  2. THIP and isoguvacine are partial agonists of GABA-stimulated benzodiazepine receptor binding.

    Science.gov (United States)

    Karobath, M; Lippitsch, M

    1979-10-15

    The effects of THIP and isoguvacine on 3H-flunitrazepam binding to washed membranes prepared from the cerebral cortex of adult rats have been examined. THIP, which has only minimal stimulatory effects on benzodiazepine (BZ) receptor binding, has been found to inhibit the stimulation induced by small concentrations (2 microM) of exogenous GABA. While isoguvacine stimulates BZ receptor binding, although to a smaller extent than GABA, it also antagonizes the stimulation of BZ receptor binding induced by GABA. Thus THIP and isoguvacine exhibit the properties of a partial agonist of GABA-stimulated BZ receptor binding.

  3. Autologous antibodies that bind neuroblastoma cells.

    Science.gov (United States)

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  4. Rescue of ligand binding of a mutant IGF-I receptor by complementation

    DEFF Research Database (Denmark)

    Chakravarty, Arjun Anders; Hinrichsen, Jane; Whittaker, Linda;

    2005-01-01

    The IGF-I receptor binds IGF-I with complex kinetics characterized by a curvilinear Scatchard plot, suggesting receptor heterogeneity and apparent negative cooperativity. To explore the molecular mechanisms underlying these properties, we have characterized the binding of a hybrid receptor formed...

  5. Hormone Binding to Recombinant Estrogen Receptors from Human, Alligator, Quail, Salamander, and Fathead Minnow

    Science.gov (United States)

    In this work, a 96-well plate estrogen receptor binding assay was developed to facilitate the direct comparison of chemical binding to full-length recombinant estrogen receptors across vertebrate classes. Receptors were generated in a baculovirus expression system. This approach ...

  6. Structural determinants for binding to angiotensin converting enzyme 2 (ACE2 and angiotensin receptors

    Directory of Open Access Journals (Sweden)

    Daniel eClayton

    2015-01-01

    Full Text Available Angiotensin converting enzyme 2 (ACE2 is a zinc carboxypeptidase involved in the renin angiotensin system (RAS and inactivates the potent vasopressive peptide angiotensin II (Ang II by removing the C-terminal phenylalanine residue to yield Ang1-7. This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule at the Mas receptor and potentially other receptors. Given the growing complexity of RAS and level of cross-talk between ligands and their corresponding enzymes and receptors, the design of molecules with selectivity for the major RAS binding partners to control cardiovascular tone is an on-going challenge. In previous studies we used single β-amino acid substitutions to modulate the structure of Ang II and its selectivity for ACE2, AT1R and angiotensin type 2 (AT2R receptor. We showed that modification at the C-terminus of Ang II generally resulted in more pronounced changes to secondary structure and ligand binding, and here we further explore this region for the potential to modulate ligand specificity. In this study, 1 a library of forty-seven peptides derived from the C-terminal tetra-peptide sequence (-IHPF of Ang II was synthesised and assessed for ACE2 binding, 2 the terminal group requirements for high affinity ACE2 binding were explored by and N- and C-terminal modification, 3 high affinity ACE2 binding chimeric AngII analogues were then synthesized and assessed, 4 the structure of the full-length Ang II analogues were assessed by circular dichroism, and 5 the Ang II analogues were assessed for AT1R/AT2R selectivity by cell-based assays. Studies on the C-terminus of Ang II demonstrated varied specificity at different residue positions for ACE2 binding and four Ang II chimeric peptides were identified as selective ligands for the AT2 receptor. Overall, these results provide insight into the residue and structural requirements for ACE2 binding and angiotensin receptor

  7. A Complete Backbone Assignment of the Apolipoprotein E LDL Receptor Binding Domain [Letter to the Editor

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Chao; Sivashanmugam, Arun; Hoyt, David W.; Wang, Jianjun

    2005-06-01

    Human apolipoprotein E (apoE) is a 299-residue exchangeable apolipoprotein that was initially recognized as a major determinant in lipoprotein metabolism and cardiovascular diseases. Recent evidence has indicated that apoE also plays critical roles in several other important biological processes not directly related to its lipid transport function, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and possibly even infectious diseases. ApoE contains two structural/functional domains: A N-terminal domain spanning residues 1-191 that is responsible for apoE's LDL receptor binding activity and a C-terminal domain (residues 216-199) that is responsible for lipoprotein-binding (1). The x-ray crystal structure of the lipid-free apoE N-terminal domain was solved by Wilson et al in 1991 which represented the only high-resolution structure of this protein. This structure showed an unusually elongated four-helix bundle (2) that was organized in such 2 a way that its hydrophobic faces were directed towards the protein interior, whereas the hydrophilic faces were oriented towards the solvent. The major receptor-binding region, residues 130-150, was located on the fourth helix. The amphipathic a-helices were connected by short loops, giving rise to a compact, globular structure. However, this structure only contained residues 23-165. Recent studies have shown that residues beyond residues 23-165 are also very important to the apoE LDL receptor binding activity. For example, a mutation at position R172 reduces the receptor binding activity of apoE to only {approx}2% (3). In addition, an E3K mutant significantly increased the apoE receptor binding activity as well (4). While the x-ray crystal structure of the apoE N-terminal domain provided detailed structural information for most region of this domain, this structure does not provide an explanation of the above experimental results regarding the structural contribution to apo

  8. Receptor binding properties of human and animal H1 influenza virus isolates.

    Science.gov (United States)

    Rogers, G N; D'Souza, B L

    1989-11-01

    -containing oligosaccharides. However, unlike the SA alpha 2,6Gal-specific H3 isolates, binding to cell surface receptors by the H1 influenza viruses is not sensitive to inhibition by horse serum glycoproteins, regardless of their receptor specificity. These results suggest that, while the H1 and H3 hemagglutinins appear to be subject to similar host-derived selective pressures, there appear to be certain fundamental differences in the detailed molecular interaction of the two hemagglutinins with their sialyloligosaccharide receptor determinants.

  9. HAMLET binding to α-actinin facilitates tumor cell detachment.

    Science.gov (United States)

    Trulsson, Maria; Yu, Hao; Gisselsson, Lennart; Chao, Yinxia; Urbano, Alexander; Aits, Sonja; Mossberg, Ann-Kristin; Svanborg, Catharina

    2011-03-08

    Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.

  10. S cysteine-rich (SCR) binding domain analysis of the Brassica self-incompatibility S-locus receptor kinase.

    Science.gov (United States)

    Kemp, Benjamin P; Doughty, James

    2007-01-01

    Brassica self-incompatibility, a highly discriminating outbreeding mechanism, has become a paradigm for the study of plant cell-cell communications. When self-pollen lands on a stigma, the male ligand S cysteine-rich (SCR), which is present in the pollen coat, is transmitted to the female receptor, S-locus receptor kinase (SRK). SRK is a membrane-spanning serine/threonine receptor kinase present in the stigmatic papillar cell membrane. Haplotype-specific binding of SCR to SRK brings about pollen rejection. The extracellular receptor domain of SRK (eSRK) is responsible for binding SCR. Based on sequence homology, eSRK can be divided into three subdomains: B lectin-like, hypervariable, and PAN. Biochemical analysis of these subdomains showed that the hypervariable subdomain is responsible for most of the SCR binding capacity of eSRK, whereas the B lectin-like and PAN domains have little, if any, affinity for SCR. Fine mapping of the SCR binding region of SRK using a peptide array revealed a region of the hypervariable subdomain that plays a key role in binding the SCR molecule. We show that residues within the hypervariable subdomain define SRK binding and are likely to be involved in defining haplotype specificity.

  11. Coregulator control of androgen receptor action by a novel nuclear receptor-binding motif.

    Science.gov (United States)

    Jehle, Katja; Cato, Laura; Neeb, Antje; Muhle-Goll, Claudia; Jung, Nicole; Smith, Emmanuel W; Buzon, Victor; Carbó, Laia R; Estébanez-Perpiñá, Eva; Schmitz, Katja; Fruk, Ljiljana; Luy, Burkhard; Chen, Yu; Cox, Marc B; Bräse, Stefan; Brown, Myles; Cato, Andrew C B

    2014-03-28

    The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

  12. Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Nuemket, Nipawan [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Yoshikazu [Creative Research Institution ' Sousei,' Hokkaido University, Sapporo 001-0021 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tsukamoto, Kentaro; Tsuji, Takao [Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192 (Japan); Nakamura, Keiji; Kozaki, Shunji [Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531 (Japan); Yao, Min [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Isao, E-mail: tanaka@castor.sci.hokudai.ac.jp [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan)

    2011-07-29

    Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

  13. Role of receptor-attached phosphates in binding of visual and non-visual arrestins to G protein-coupled receptors.

    Science.gov (United States)

    Gimenez, Luis E; Kook, Seunghyi; Vishnivetskiy, Sergey A; Ahmed, M Rafiuddin; Gurevich, Eugenia V; Gurevich, Vsevolod V

    2012-03-16

    Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in β-strand I was shown to disrupt the interaction of α-helix I, β-strand I, and the C-tail of visual arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins in which the K2A mutations had little effect on net BRET(max) values for the M2 muscarinic acetylcholine (M2R), β(2)-adrenergic (β(2)AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual arrestins normally, whereas phosphorylation-deficient β(2)AR mutants bound arrestins at 20-50% of the level of wild type β(2)AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. Although arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of arrestin-2 and -3 is much greater in the case of β(2)AR than M2R and D2 dopamine receptor.

  14. Receptor-Like Function of Heparin in the Binding and Uptake of Neutral Lipids

    Science.gov (United States)

    Bosner, Matthew S.; Gulick, Tod; Riley, D. J. S.; Spilburg, Curtis A.; Lange, Louis G.

    1988-10-01

    Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids (e.g., cholesteryl esters, triglycerides, and fatty acids) are well studied, but the details of their movement from a binding compartment to a metabolic compartment deserve further attention. Since all neutral lipids must cross hydrophilic segments of plasma membranes during such movement, we postulate that a critical receptor-like site exists on the plasma membrane to mediate a step between binding and metabolism and that membrane-associated heparin is a key part of this mediator. For example, intestinal brush border membranes containing heparin bind homogeneous human pancreatic 125I-labeled cholesterol esterase (100 kDa) and 125I-labeled triglyceride lipase (52 kDa). This interaction is enzyme concentration-dependent, specific, and saturable and is reversed upon addition of soluble heparin. Scatchard analysis demonstrates a single class of receptors with a Kd of 100 nM and a Bmax of approximately 50-60 pmol per mg of vesicle protein. In contrast, enzymes associated with the hydrolysis of hydrophilic compounds such as amylase, phospholipase A2, and deoxyribonuclease do not bind to intestinal membranes in this manner. Human pancreatic cholesterol esterase also binds specifically and saturably to cultured intestinal epithelial cells (CaCo-2), and soluble heparin significantly diminishes the cellular uptake of the resultant hydrophobic reaction products (cholesterol and free fatty acids). We conclude that a physiological role for intestinal heparin is that of a mediator to bind neutral lipolytic enzymes at the brush border and thus promote absorption of the subsequent hydrolyzed nutrients in the intestine. This mechanism may be a generalizable pathway for transport of neutral lipids into endothelial and other cells.

  15. Mapping Substance P Binding Sites on the Neurokinin-1 Receptor Using Genetic Incorporation of a Photoreactive Amino Acid

    DEFF Research Database (Denmark)

    Valentin-Hansen, Louise; Park, Minyoung; Huber, Thomas;

    2014-01-01

    Substance P (SP) is a neuropeptide that mediates numerous physiological responses, including transmission of pain and inflammation through the neurokinin-1 (NK1) receptor, a G protein-coupled receptor. Previous mutagenesis studies and photoaffinity labeling using ligand analogues suggested that t...... possess multiple determinants for SP binding and demonstrate the utility of genetically encoded photocross-linking to map complex multitopic binding sites on G protein-coupled receptors in a cell-based assay format....... that the binding site for SP includes multiple domains in the N-terminal (Nt) segment and the second extracellular loop (ECLII) of NK1. To map precisely the NK1 residues that interact with SP, we applied a novel receptor-based targeted photocross-linking approach. We used amber codon suppression to introduce...

  16. Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1.

    Science.gov (United States)

    Xu, Huifang; Raynal, Nicolas; Stathopoulos, Stavros; Myllyharju, Johanna; Farndale, Richard W; Leitinger, Birgit

    2011-01-01

    The discoidin domain receptors, DDR1 and DDR2 are cell surface receptor tyrosine kinases that are activated by triple-helical collagen. While normal DDR signalling regulates fundamental cellular processes, aberrant DDR signalling is associated with several human diseases. We previously identified GVMGFO (O is hydroxyproline) as a major DDR2 binding site in collagens I-III, and located two additional DDR2 binding sites in collagen II. Here we extend these studies to the homologous DDR1 and the identification of DDR binding sites on collagen III. Using sets of overlapping triple-helical peptides, the Collagen II and Collagen III Toolkits, we located several DDR2 binding sites on both collagens. The interaction of DDR1 with Toolkit peptides was more restricted, with DDR1 mainly binding to peptides containing the GVMGFO motif. Triple-helical peptides containing the GVMGFO motif induced DDR1 transmembrane signalling, and DDR1 binding and receptor activation occurred with the same amino acid requirements as previously defined for DDR2. While both DDRs exhibit the same specificity for binding the GVMGFO motif, which is present only in fibrillar collagens, the two receptors display distinct preferences for certain non-fibrillar collagens, with the basement membrane collagen IV being exclusively recognised by DDR1. Based on our recent crystal structure of a DDR2-collagen complex, we designed mutations to identify the molecular determinants for DDR1 binding to collagen IV. By replacing five amino acids in DDR2 with the corresponding DDR1 residues we were able to create a DDR2 construct that could function as a collagen IV receptor.

  17. The Sigma-2 Receptor and Progesterone Receptor Membrane Component 1 are Different Binding Sites Derived From Independent Genes

    Directory of Open Access Journals (Sweden)

    Uyen B. Chu

    2015-11-01

    Full Text Available The sigma-2 receptor (S2R is a potential therapeutic target for cancer and neuronal diseases. However, the identity of the S2R has remained a matter of debate. Historically, the S2R has been defined as (1 a binding site with high affinity to 1,3-di-o-tolylguanidine (DTG and haloperidol but not to the selective sigma-1 receptor ligand (+-pentazocine, and (2 a protein of 18–21 kDa, as shown by specific photolabeling with [3H]-Azido-DTG and [125I]-iodoazido-fenpropimorph ([125I]-IAF. Recently, the progesterone receptor membrane component 1 (PGRMC1, a 25 kDa protein, was reported to be the S2R (Nature Communications, 2011, 2:380. To confirm this identification, we created PGRMC1 knockout NSC34 cell lines using the CRISPR/Cas9 technology. We found that in NSC34 cells devoid of or overexpressing PGRMC1, the maximum [3H]-DTG binding to the S2R (Bmax as well as the DTG-protectable [125I]-IAF photolabeling of the S2R were similar to those of wild-type control cells. Furthermore, the affinities of DTG and haloperidol for PGRMC1 (KI = 472 μM and 350 μM, respectively, as determined in competition with [3H]-progesterone, were more than 3 orders of magnitude lower than those reported for the S2R (20–80 nM. These results clarify that PGRMC1 and the S2R are distinct binding sites expressed by different genes.

  18. T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors

    DEFF Research Database (Denmark)

    Jamnani, Fatemeh Rahimi; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali;

    2014-01-01

    Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination...

  19. Efficient and cost-effective experimental determination of kinetic constants and data: the success of a Bayesian systematic approach to drug transport, receptor binding, continuous culture and cell transport kinetics.

    Science.gov (United States)

    Murphy, Emma F; Gilmour, Steven G; Crabbe, M James C

    2004-01-02

    Details about the parameters of kinetic systems are crucial for progress in both medical and industrial research, including drug development, clinical diagnosis and biotechnology applications. Such details must be collected by a series of kinetic experiments and investigations. The correct design of the experiment is essential to collecting data suitable for analysis, modelling and deriving the correct information. We have developed a systematic and iterative Bayesian method and sets of rules for the design of enzyme kinetic experiments. Our method selects the optimum design to collect data suitable for accurate modelling and analysis and minimises the error in the parameters estimated. The rules select features of the design such as the substrate range and the number of measurements. We show here that this method can be directly applied to the study of other important kinetic systems, including drug transport, receptor binding, microbial culture and cell transport kinetics. It is possible to reduce the errors in the estimated parameters and, most importantly, increase the efficiency and cost-effectiveness by reducing the necessary amount of experiments and data points measured.

  20. Molecular cell biology of androgen receptor signalling.

    Science.gov (United States)

    Bennett, Nigel C; Gardiner, Robert A; Hooper, John D; Johnson, David W; Gobe, Glenda C

    2010-06-01

    The classical action of androgen receptor (AR) is to regulate gene transcriptional processes via AR nuclear translocation, response element binding and recruitment of, or crosstalk with, transcription factors. AR also utilises non-classical, non-genomic mechanisms of signal transduction. These precede gene transcription or protein synthesis, and involve steroid-induced modulation of cytoplasmic or cell membrane-bound regulatory proteins. Despite many decades of investigation, the role of AR in gene regulation of cells and tissues remains only partially characterised. AR exerts most of its effects in sex hormone-dependent tissues of the body, but the receptor is also expressed in many tissues not previously thought to be androgen sensitive. Thus it is likely that a complex, more over-arching, role for AR exists. Each AR domain co-ordinates a multitude of individual and vital roles via a diverse array of interacting partner molecules that are necessary for cellular and tissue development and maintenance. Aberrant AR activity, promoted by mutations or binding partner misregulation, can present as many clinical manifestations including androgen insensitivity syndrome and prostate cancer. In the case of malignant prostate cancer, treatment generally revolves around androgen deprivation therapies designed to interfere with AR action and the androgen signalling axis. Androgen therapies for prostate cancer often fail, highlighting a real need for increased research into AR function.

  1. [Regulation of G protein-coupled receptor function by its binding proteins].

    Science.gov (United States)

    Nakahata, Norimichi; Saito, Masaki

    2007-01-01

    G protein-coupled receptors (GPCRs) are seven transmembrane receptors with an N-terminus in the extracellular region and C-terminus in the intracellular region. When an agonist binds to a GPCR, a signal is transduced into a cell through the activation of trimeric G proteins. Recently, it has been shown that the activities of GPCRs are regulated by multiple mechanisms. One of the mechanisms is regulation through the binding proteins to the carboxy (C)-terminus of GPCRs. In the present study, the binding partners for the C-terminus of the parathyroid hormone receptor (PTHR) and thromboxane A(2) receptor (TP) were searched for using yeast two-hybrid screening, and the functions of these proteins were investigated. We identified t-complex testis expressed-1 (Tctex-1) and 4.1G as associated proteins for the PTHR. Tctex-1 is one of the light chains of cytoplasmic dynein, which is a motor protein across microtubles. We found that Tctex-1 was involved in agonist-induced internalization of the PTHR. 4.1G, a cytoskeletal protein, facilitated the cell surface localization of the PTHR and augmented PHTR-mediated signal transduction. TPs consists of two splicing variants, TPalpha and TPbeta. As a result of yeast two-hybrid screening, two proteasomal proteins, proteasome activator PA28gamma and proteasome subunit alpha7, were identified as direct interacting proteins for TPbeta. TPbeta has a tendency to be retained in the intracellular compartment, probably due to its binding to proteasomes. We also demonstrated that TPalpha and TPbeta formed heterodimers, and the signal transduction through TPalpha was reduced by the formation of heterodimers. In conclusion, the proteins bound to GPCRs may regulate the intracellular traffic of GPCRs.

  2. Binding of estrogen receptor with estrogen conjugated to bovine serum albumin (BSA).

    Science.gov (United States)

    Taguchi, Yasuto; Koslowski, Mirek; Bodenner, Donald L

    2004-08-19

    BACKGROUND: The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E2-BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E2-BSA does not compete for E2 binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E2 and E2-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E2-BSA preparations with unconjugated E2 (usually between 3-5%), the latter was carefully removed by ultrafiltration. RESULTS: As previously reported, a 10-to 1000-fold molar excess of E2-BSA was unable to compete with 3H-E2 binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E2-BSA, the binding of 3H-E2 was significantly reduced. E2-BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E2-BSA (E2-BSA-FITC). Staining was restricted to the cell membrane when E2-BSA-FITC was incubated with stable transfectants of the murine ERalpha within ER-negative HeLa cells and with MC7 cells that endogenously produce ERalpha. This staining appeared highly specific since it was competed by pre-incubation with E2 in a dose dependent manner and with the competitor ICI-182,780. CONCLUSIONS: These results demonstrate that E2-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E2-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E2. More importantly, these findings demonstrate

  3. Cloning, expression, and ligand-binding characterization of two neuropeptide Y receptor subtypes in orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Wang, Fei; Chen, Weimin; Lin, Haoran; Li, Wensheng

    2014-12-01

    As one of the most important multifunctional peptides, neuropeptide Y (NPY) performs its physiological functions through different subtype receptors. In this study, full-length cDNAs of two NPY receptors (YRs) in orange-spotted grouper (Epinephelus coioides) were cloned and named npy8br (y8b) and npy2r (y2). Phylogenetic analysis indicated that the Y8b receptor is an ortholog of the teleostean Y8b receptor, which belongs to the Y1 subfamily, and the Y2 receptor is an ortholog of the teleostean Y2 receptor, which belongs to the Y2 subfamily. Both of the YRs have G protein-coupled receptor family profiles. Multiple alignments demonstrate that the extracellular loop regions of YRs have distinctive residues of each species. Expression profile analysis revealed that the grouper Y8b receptor mRNA is primarily expressed in the brain, stomach and intestine, while the grouper Y2 receptor mRNA is primarily expressed in the brain, ovary, liver and heart. Double immunofluorescence analysis determined that the grouper YRs interact with the grouper NPY around the human embryonic kidney 293T cell surface. Furthermore, site-directed mutagenesis in a phage display system revealed that Asp(6.59) might be a common NPY-binding site, while Asp(2.68) of the Y8b receptor and Glu(5.24) of the Y2 receptor could be likely involved in subtype-specific binding. Combining the expression profile and ligand-binding feature, the grouper Y8b receptor could be involved in regulating food intake via the brain-gut axis and the grouper Y2 receptor might play a role in balancing the regulatory activity of the Y8b receptor and participate in metabolism in the liver and ovary.

  4. Tumor necrosis factor alpha and interleukin 11 secreted by malignant breast epithelial cells inhibit adipocyte differentiation by selectively down-regulating CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma: mechanism of desmoplastic reaction.

    Science.gov (United States)

    Meng, L; Zhou, J; Sasano, H; Suzuki, T; Zeitoun, K M; Bulun, S E

    2001-03-01

    The dense layer of fibroblasts that accumulate around malignant breast epithelial cells (i.e., desmoplastic reaction) arises from the breast adipose tissue and provides structural and biochemical support for breast cancer. We report herein a number of epithelial-stromal interactions responsible for desmoplastic reaction in breast cancer using cultured 3T3-L1 murine fibroblasts and human adipose fibroblasts, which can be activated with a mixture of hormones to differentiate to mature adipocytes. Adipocyte differentiation was inhibited by coculturing fibroblasts with various breast cancer cell lines (T47D, MCF-7, SSC202, SSC78, and SSC30) completely or by breast cancer cell conditioned media in a dose-dependent manner; on the other hand, adipocyte differentiation was not inhibited by coculturing with normal human primary mammary epithelial cell conditioned medium. This tumor effect was eliminated using neutralizing antibodies against tumor necrosis factor (TNF)-alpha or interleukin (IL)-11. TNF-alpha and IL-11 levels were 2.5-3 times higher in T47D conditioned medium compared with control medium, and TNF-alpha transcripts were detectable in T47D but not in 3T3-L1 cells in culture, indicating that the malignant epithelial cell is the major site of cytokine production. This was confirmed in vivo in mastectomy specimens, where immunoreactive TNF-alpha and IL-11 were readily detectable in malignant epithelial cells but not in the majority of the surrounding fibroblasts. Adipocyte differentiation is mediated by the expression of a cascade of adipogenic transcription factors, including CCAAT/enhancer binding protein (C/EBP)beta, C/EBPdelta, peroxisome proliferator-activated receptor (PPAR)gamma and C/EBPalpha. C/EBPalpha and PPARgamma are essential for this process. We demonstrated by Northern analysis that exposure of activated 3T3-L1 cells to T47D cell conditioned medium strikingly decreased the levels of PPARgamma and C/EBPalpha transcripts and increased the levels of C

  5. Why does the agonist [{sup 18}F]FP-TZTP bind preferentially to the M{sub 2} muscarinic receptor?

    Energy Technology Data Exchange (ETDEWEB)

    Ravasi, L. [National Institutes of Health, PET Radiochemistry Group, National Institute for Biomedical Imaging and Bioengineering, Bethesda, MD (United States); Universita degli Studi di Milano, Institute of Radiological Sciences, Milan (Italy); Kiesewetter, D.O.; Shimoji, K. [National Institutes of Health, PET Radiochemistry Group, National Institute for Biomedical Imaging and Bioengineering, Bethesda, MD (United States); Lucignani, G. [Universita degli Studi di Milano, Institute of Radiological Sciences, Milan (Italy); European Institute of Oncology, Unit of Molecular Imaging, Division of Radiation Therapy, Milan (Italy); Eckelman, W.C. [National Institutes of Health, PET Department, Clinical Center, Bethesda, MD (United States); Molecular Tracer LLC, Bethesda, MD (United States)

    2006-03-15

    Preferential binding of FP-TZTP at the M{sub 2} receptor in vivo led to investigation of [{sup 18}F]FP-TZTP as a potential PET tracer for Alzheimer's disease, in which a substantial reduction of M{sub 2} receptors has been observed in autopsy studies. We hereby investigated in vitro the FP-TZTP behavior to further elucidate the properties of FP-TZTP that lead to its M{sub 2} selectivity. Chinese hamster ovarian cells expressing the five subtypes of human muscarinic receptor as well as the wild type were harvested in culture to assess equilibrium binding. Specific binding was calculated by subtraction of non-specific binding from total binding. Internal specific binding was calculated by subtraction of external specific binding from the total specific binding. Saturation assays were also performed to calculate B{sub max}, K{sub i}, and IC{sub 50}. In addition, equilibrium binding and dissociation kinetic studies were performed on rat brain tissue. Selected regions of interest were drawn on the digital autoradiograms and [{sup 18}F]FP-TZTP off-rates were determined by measurement of the rate of release into a buffer solution of [{sup 18}F]FP-TZTP from slide-bound cells that had been preincubated with [{sup 18}F]FP-TZTP. At equilibrium in vitro, M{sub 2} subtype selectivity of [{sup 18}F]FP-TZTP was not evident. We demonstrated that ATP-dependent mechanisms are not responsible for FP-TZTP M{sub 2} selectivity. In vitro off-rate studies from rat brain tissue showed that the off-rate of FP-TZTP varied with the percentage of M{sub 2} subtype in the tissue region. The slower dissociation kinetics of FP-TZTP from M{sub 2} receptors compared with the four other muscarinic receptor subtypes may be a factor in its M{sub 2} selectivity. (orig.)

  6. Changes in 5-HT4 receptor and 5-HT transporter binding in olfactory bulbectomized and glucocorticoid receptor heterozygous mice

    DEFF Research Database (Denmark)

    Licht, Cecilie L; Kirkegaard, Lisbeth; Zueger, Maha;

    2010-01-01

    . The olfactory bulbectomized mice displayed increased activity in the open field test, a characteristic depression-like feature of this model. After bulbectomy, 5-HT(4) receptor binding was increased in the ventral hippocampus (12%) but unchanged in the dorsal hippocampus, frontal and caudal caudate putamen....... Among post hoc analyzed regions, there was a 14% decrease in 5-HT(4) receptor binding in the olfactory tubercles. The 5-HTT binding was unchanged in the hippocampus and caudate putamen of bulbectomized mice but post hoc analysis showed small decreases in lateral septum and lateral globus pallidus....... In comparison, GR(+/-) mice had increased 5-HT(4) receptor (11%) binding in the caudal caudate putamen and decreased 5-HTT binding in the frontal caudate putamen but no changes in dorsal and ventral hippocampus. Post hoc analysis showed increased 5-HT(4) receptor binding in the olfactory tubercles of GR...

  7. The MHC class I binding proteins LIR-1 and LIR-2 inhibit Fc receptor-mediated signaling in monocytes.

    Science.gov (United States)

    Fanger, N A; Cosman, D; Peterson, L; Braddy, S C; Maliszewski, C R; Borges, L

    1998-11-01

    The MHC class I binding proteins leukocyte immunoglobulin-like receptor (LIR)-1 and -2 recognize a similar broad spectrum of HLA-A, -B and -C alleles but are differentially expressed in lymphocytes, monocytes, and dendritic cells. In monocytes, phosphorylation of LIR-1 and LIR-2 results in the binding of the tyrosine phosphatase SHP-1. Coligation of either LIR with Fcgamma receptor I (CD64) inhibits tyrosine phosphorylation of the associated Fc receptor gamma chain and Syk molecules, as well as intracellular calcium mobilization. These findings suggest that LIR-1 and LIR-2 function as unique MHC class I receptors involved in the inhibition or down-modulation of monocyte activation signals, particularly those mediated through the receptors for IgG, IgE and IgA.

  8. Toll-like receptor 2 and nucleotide-binding oligomerization domain-2 play divergent roles in the recognition of gut-derived lactobacilli and bifidobacteria in dendritic cells

    DEFF Research Database (Denmark)

    Zeuthen, Louise; Fink, Lisbeth Nielsen; Frøkiær, Hanne

    2008-01-01

    -marrow-derived DC lacking NOD2 produce higher levels of interleukin-10 (IL-10) and reduced levels of IL-12 and tumour necrosis factor-[alpha] (TNF-[alpha]) in response to LAB. This indicates that peptidoglycan is partly responsible for the T helper type 1 skewing effect of certain LAB. Dendritic cells that are TLR2......-/- produce less IL-12 and TNF-[alpha] and more IL-10 in response to some strains of lactobacilli, while they produce more IL-12 and less IL-10 in response to bifidobacteria. The same tendency was found in human monocyte-derived DC. We have previously reported that the weak IL-12-inducing and TNF......-[alpha]-inducing bifidobacteria inhibit the T helper type 1 skewing effect induced by strong immunostimulatory lactobacilli. Here we show that this immunoinhibitory effect of bifidobacteria is dependent on TLR2 and independent of NOD2. Moreover, independently of the cytokine pattern induced by intact LAB, cell wall fractions...

  9. The Role of Nucleotide-Binding Oligomerization Domain-Like Receptors in Pulmonary Infection.

    Science.gov (United States)

    Wiese, Kristin M; Coates, Bria M; Ridge, Karen M

    2017-08-01

    Pneumonia is caused by both viral and bacterial pathogens and is responsible for a significant health burden in the Unites States. The innate immune system is the human body's first line of defense against these pathogens. The recognition of invading pathogens via pattern recognition receptors leads to proinflammatory cytokine and chemokine production, followed by recruitment and activation of effector immune cells. The nonspecific inflammatory nature of the innate immune response can result in immunopathology that is detrimental to the host. In this review, we focus on one class of pattern recognition receptors, the nucleotide-binding oligomerization domain (NOD)-like receptors, specifically NOD1 and NOD2, and their role in host defense against viral and bacterial pathogens of the lung, including influenza, respiratory syncytial virus, Streptococcus pneumoniae, Chlamydophila pneumoniae, and Staphylococcus aureus. It is hoped that improved understanding of NOD1 and NOD2 activity in pneumonia will facilitate the development of novel therapies and promote improved patient outcomes.

  10. A novel phage-library-selected peptide inhibits human TNF-α binding to its receptors.

    Science.gov (United States)

    Brunetti, Jlenia; Lelli, Barbara; Scali, Silvia; Falciani, Chiara; Bracci, Luisa; Pini, Alessandro

    2014-06-03

    We report the identification of a new human tumor necrosis factor-alpha (TNF-α) specific peptide selected by competitive panning of a phage library. Competitive elution of phages was obtained using the monoclonal antibody adalimumab, which neutralizes pro-inflammatory processes caused by over-production of TNF-α in vivo, and is used to treat severe symptoms of rheumatoid arthritis. The selected peptide was synthesized in monomeric and branched form and analyzed for binding to TNF-α and competition with adalimumab and TNF-α receptors. Results of competition with TNF-α receptors in surface plasmon resonance and melanoma cells expressing both TNF receptors make the peptide a candidate compound for the development of a novel anti-TNF-α drug.

  11. Aptamers Binding to c-Met Inhibiting Tumor Cell Migration.

    Directory of Open Access Journals (Sweden)

    Birgit Piater

    Full Text Available The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF. Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX. CLN64 and a previously described single-stranded DNA (ssDNA aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.

  12. Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J;

    1995-01-01

    We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA...

  13. MANAGING TIGHT BINDING RECEPTORS FOR NEW SPEARATIONS TECHNOLOGIES

    Energy Technology Data Exchange (ETDEWEB)

    DARYLE H BUSCH RICHARD S GIVENS

    2004-12-10

    even more interesting. They convert from rings to structures that wrap around a metal ion to form a cage. These ligands are called cryptands. Switch release is accomplished by photolytic cleavage of a bond to convert a cyclic ligand into a linear ligand or to break similar bonds in a cryptate. Our studies have demonstrated switch binding and switch release with cryptates of calcium. These remarkable cyclic ligands and cage-like ligands are indeed tight-binding and may, in principle, be incorporated in various separations methodologies, including the soil poultice. The soil poultice mimics the way in which microbes secrete extremely powerful ligands into the soil in order to harvest iron. The cellular membrane of the microbe recognizes the iron/ligand complex and admits it into the cell. The soil poultice uses molecularly imprinted polymers (MIPs) to play the role of the cellular membrane. Imprinting involves creation of the polymer in the presence of the metal/ligand complex. In principle, a well design ligand/MIP combination can be highly selective toward almost any targeted metal ion. The principles for that design are the focus of these investigations. An imprinting molecule can interact with the polymer through any, some, or all of the so-called supramolecular modes; e.g., hydrogen bonding, electrostatic charge, minor ligand bonding, Pi-Pi stacking, and hydrophobic and van der Waals interactions. Historically these modes of binding have given MIPs only small re-binding capacities and very limited selectivities. This program has shown that each mode of interaction can be made more powerful than previously suspected and that combinations of different supramolecular interaction modes can produce remarkable synergisms. The results of this systematic study provide a firm foundation for tailoring molecular imprinted polymers for reclamation of specific metal ion, including those important to the DOE EM mission.

  14. Receptor specificity and erythrocyte binding preferences of avian influenza viruses isolated from India

    Directory of Open Access Journals (Sweden)

    Pawar Shailesh D

    2012-10-01

    Full Text Available Abstract Introduction Hemagglutination (HA and hemagglutination inhibition (HI assays are conventionally used for detection and identification of influenza viruses. HI assay is also used for detection of antibodies against influenza viruses. Primarily turkey or chicken erythrocytes [red blood cells (RBCs] are used in these assays, as they are large, nucleated, and sediment fast, which makes it easy to determine the titer. Human influenza viruses agglutinate RBCs from chicken, human, and guinea pig, but not from horse. Human influenza viruses bind preferentially to sialic acid (SA linked to galactose (Gal by α 2, 6 linkage (SA α 2, 6-Gal, whereas avian influenza (AI viruses bind preferentially to SA α 2, 3-Gal linkages. With this background, the present study was undertaken to study erythrocyte binding preferences and receptor specificities of AI viruses isolated from India. Materials and methods A total of nine AI virus isolates (four subtypes from India and three reference AI strains (three subtypes were tested in HA and HI assays against mammalian and avian erythrocytes. The erythrocytes from turkey, chicken, goose, guinea pig and horse were used in the study. The receptor specificity determination assays were performed using goose and turkey RBCs. The amino acids present at 190 helix, 130 and 220 loops of the receptor-binding domain of the hemagglutinin protein were analyzed to correlate amino acid changes with the receptor specificity. Results All tested highly pathogenic avian influenza (HPAI H5N1 viruses reacted with all five types of RBCs in the HA assay; AI H9N2 and H5N2 viruses did not react with horse RBCs. For H5N1 viruses guinea pig and goose RBCs were best for both HA and HI assays. For H9N2 viruses, guinea pig, fowl and turkey RBCs were suitable. For other tested AI subtypes, avian and guinea pig RBCs were better. Eight isolates of H5N1, one H4N6 and one H7N1 virus showed preference to avian sialic acid receptors. Importantly

  15. Binding characteristics of brain-derived neurotrophic factor to its receptors on neurons from the chick embryo

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Tebar, A.; Barde, Y.A.

    1988-09-01

    Brain-derived neurotrophic factor (BDNF), a protein known to support the survival of embryonic sensory neurons and retinal ganglion cells, was derivatized with 125I-Bolton-Hunter reagent and obtained in a biologically active, radioactive form (125I-BDNF). Using dorsal root ganglion neurons from chick embryos at 9 d of development, the basic physicochemical parameters of the binding of 125I-BDNF with its receptors were established. Two different classes of receptors were found, with dissociation constants of 1.7 x 10(-11) M (high-affinity receptors) and 1.3 x 10(-9) M (low-affinity receptors). Unlabeled BDNF competed with 125I-BDNF for binding to the high-affinity receptors with an inhibition constant essentially identical to the dissociation constant of the labeled protein: 1.2 x 10(-11) M. The association and dissociation rates from both types of receptors were also determined, and the dissociation constants calculated from these kinetic experiments were found to correspond to the results obtained from steady-state binding. The number of high-affinity receptors (a few hundred per cell soma) was 15 times lower than that of low-affinity receptors. No high-affinity receptors were found on sympathetic neurons, known not to respond to BDNF, although specific binding of 125I-BDNF to these cells was detected at a high concentration of the radioligand. These results are discussed and compared with those obtained with nerve growth factor on the same neuronal populations.

  16. The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake

    DEFF Research Database (Denmark)

    Hvidberg, Vibeke; Jacobsen, Christian; Strong, Roland K

    2005-01-01

    in delivering iron to cells during formation of the tubular epithelial cells of the primordial kidney. No cellular receptor for NGAL has been described. We show here that megalin, a member of the low-density lipoprotein receptor family expressed in polarized epithelia, binds NGAL with high affinity, as shown...

  17. Identification and Preparation of a Novel Chemokine Receptor-Binding Domain in the Cytoplasmic Regulator FROUNT.

    Science.gov (United States)

    Sonoda, Akihiro; Yoshinaga, Sosuke; Yunoki, Kaori; Ezaki, Soichiro; Yano, Kotaro; Takeda, Mitsuhiro; Toda, Etsuko; Terashima, Yuya; Matsushima, Kouji; Terasawa, Hiroaki

    2017-03-24

    FROUNT is a cytoplasmic protein that binds to the membrane-proximal C-terminal regions (Pro-Cs) of chemokine receptors, CCR2 and CCR5. The FROUNT-chemokine receptor interactions play a pivotal role in the migration of inflammatory immune cells, indicating the potential of FROUNT as a drug target for inflammatory diseases. To provide the foundation for drug development, structural information of the Pro-C binding region of FROUNT is desired. Here, we defined the novel structural domain (FNT-CB), which mediates the interaction with the chemokine receptors. A recombinant GST-tag-fused FNT-CB protein expression system was constructed. The protein was purified by affinity chromatography and then subjected to in-gel protease digestion of the GST-tag. The released FNT-CB was further purified by anion-exchange and size-exclusion chromatography. Purified FNT-CB adopts a helical structure, as indicated by CD. NMR line-broadening indicated that weak aggregation occurred at sub-millimolar concentrations, but the line-broadening was mitigated by using a deuterated sample in concert with transverse relaxation-optimized spectroscopy. The specific binding of FNT-CB to CCR2 Pro-C was confirmed by the fluorescence-based assay. The improved NMR spectral quality and the retained functional activity of FNT-CB support the feasibility of further structural and functional studies targeted at the anti-inflammatory drug development.

  18. A chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus.

    Science.gov (United States)

    Alejo, Alí; Ruiz-Argüello, M Begoña; Ho, Yin; Smith, Vincent P; Saraiva, Margarida; Alcami, Antonio

    2006-04-11

    Variola virus (VaV) is the causative agent of smallpox, one of the most devastating diseases encountered by man, that was eradicated in 1980. The deliberate release of VaV would have catastrophic consequences on global public health. However, the mechanisms that contribute to smallpox pathogenesis are poorly understood at the molecular level. The ability of viruses to evade the host defense mechanisms is an important determinant of viral pathogenesis. Here we show that the tumor necrosis factor receptor (TNFR) homologue CrmB encoded by VaV functions not only as a soluble decoy TNFR but also as a highly specific binding protein for several chemokines that mediate recruitment of immune cells to mucosal surfaces and the skin, sites of virus entry and viral replication at late stages of smallpox. CrmB binds chemokines through its C-terminal domain, which is unrelated to TNFRs, was named smallpox virus-encoded chemokine receptor (SECRET) domain and uncovers a family of poxvirus chemokine inhibitors. An active SECRET domain was found in another viral TNFR (CrmD) and three secreted proteins encoded by orthopoxviruses. These findings identify a previously undescribed chemokine-binding and inhibitory domain unrelated to host chemokine receptors and a mechanism of immune modulation in VaV that may influence smallpox pathogenesis.

  19. Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca2+-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus

    OpenAIRE

    Patricia eRivera; Sergio eArrabal; Manuel eCifuentes; Grondona, Jesús M; Margarita ePérez-Martín; Leticia eRubio; Antonio eVargas Fuentes; Antonia eSerrano; Francisco Javier ePavon; Juan eSuarez; Fernando eRodríguez de Fonseca

    2014-01-01

    The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca2+ and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca2+-binding proteins (CaBPs) is still unknown. For this purpose, we use...

  20. Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells

    NARCIS (Netherlands)

    Sinha, B; Francois, P; Que, Y A; Hussain, M; Heilmann, C; Moreillon, P; Lew, D; Krause, K H; Peters, Georg; Herrmann, M

    2000-01-01

    Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell.

  1. Soluble Human Intestinal Lactoferrin Receptor: Ca(2+)-Dependent Binding to Sepharose-Based Matrices.

    Science.gov (United States)

    Oshima, Yuta; Seki, Kohei; Shibuya, Masataka; Naka, Yuki; Yokoyama, Tatsuya; Sato, Atsushi

    2016-01-01

    A soluble form of human intestinal lactoferrin receptor (shLFR) is identical to human intelectin-1 (hITLN-1), a galactofuranose-binding protein that acts as a host defense against invading pathogenic microorganisms. We found that recombinant shLFR, expressed in mammalian cells (CHO DG44, COS-1, and RK13), binds tightly to Sepharose 4 Fast Flow (FF)-based matrices in a Ca(2+)-dependent manner. This binding of shLFR to Sepharose 4 FF-based matrices was inhibited by excess D-galactose, but not by D-glucose, suggesting that shLFR recognizes repeating units of α-1,6-linked D-galactose in Sepharose 4 FF. Furthermore, shLFR could bind to both Sepharose 4B- and Sepharose 6B-based matrices that were not crosslinked in a similar manner as to Sepharose 4 FF-based matrices. Therefore, shLFR (hITLN-1) binds to Sepharose-based matrices in a Ca(2+)-dependent manner. This binding property is most likely related to the ability, as host defense lectins, to recognize sepharose (agarobiose)-like structures present on the surface of invading pathogenic microorganisms.

  2. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    Science.gov (United States)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

  3. Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range

    Science.gov (United States)

    Ventura, Alejandra C.; Bush, Alan; Vasen, Gustavo; Goldín, Matías A.; Burkinshaw, Brianne; Bhattacharjee, Nirveek; Folch, Albert; Brent, Roger; Chernomoretz, Ariel; Colman-Lerner, Alejandro

    2014-01-01

    Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general “systems level” mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step. PMID:25172920

  4. Distinctive receptor binding properties of the surface glycoprotein of a natural Feline Leukemia Virus isolate with unusual disease spectrum

    Directory of Open Access Journals (Sweden)

    Albritton Lorraine M

    2011-05-01

    Full Text Available Abstract Background Feline leukemia virus (FeLV-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor. Results Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect. Conclusions The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread in vivo, or by facilitating entry into a novel target cell with a low receptor density.

  5. Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization.

    Directory of Open Access Journals (Sweden)

    Irina M Kuznetsova

    Full Text Available In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA and ANS - bovine serum albumin (BSA interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.

  6. The SARS Coronavirus S Glycoprotein Receptor Binding Domain: Fine Mapping and Functional Characterization

    Directory of Open Access Journals (Sweden)

    Xiao Xiaodong

    2005-08-01

    Full Text Available Abstract The entry of the SARS coronavirus (SCV into cells is initiated by binding of its spike envelope glycoprotein (S to a receptor, ACE2. We and others identified the receptor-binding domain (RBD by using S fragments of various lengths but all including the amino acid residue 318 and two other potential glycosylation sites. To further characterize the role of glycosylation and identify residues important for its function as an interacting partner of ACE2, we have cloned, expressed and characterized various soluble fragments of S containing RBD, and mutated all potential glycosylation sites and 32 other residues. The shortest of these fragments still able to bind the receptor ACE2 did not include residue 318 (which is a potential glycosylation site, but started at residue 319, and has only two potential glycosylation sites (residues 330 and 357. Mutation of each of these sites to either alanine or glutamine, as well as mutation of residue 318 to alanine in longer fragments resulted in the same decrease of molecular weight (by approximately 3 kDa suggesting that all glycosylation sites are functional. Simultaneous mutation of all glycosylation sites resulted in lack of expression suggesting that at least one glycosylation site (any of the three is required for expression. Glycosylation did not affect binding to ACE2. Alanine scanning mutagenesis of the fragment S319–518 resulted in the identification of ten residues (K390, R426, D429, T431, I455, N473, F483, Q492, Y494, R495 that significantly reduced binding to ACE2, and one residue (D393 that appears to increase binding. Mutation of residue T431 reduced binding by about 2-fold, and mutation of the other eight residues – by more than 10-fold. Analysis of these data and the mapping of these mutations on the recently determined crystal structure of a fragment containing the RBD complexed to ACE2 (Li, F, Li, W, Farzan, M, and Harrison, S. C., submitted suggested the existence of two hot

  7. Retinoic acid receptors recognize the mouse genome through binding elements with diverse spacing and topology.

    Science.gov (United States)

    Moutier, Emmanuel; Ye, Tao; Choukrallah, Mohamed-Amin; Urban, Sylvia; Osz, Judit; Chatagnon, Amandine; Delacroix, Laurence; Langer, Diana; Rochel, Natacha; Moras, Dino; Benoit, Gerard; Davidson, Irwin

    2012-07-27

    Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.

  8. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    2000-08-01

    Peripheral benzodiazepine receptor (PBR) agonist [(3)H]Ro5-4864 has been shown to bind with high affinity to the human breast cancer cell line BT-20. Therefore, we investigated different human breast cancer cell lines with regard to binding to [(3)H]Ro5-4864 and staining with the PBR-specific monoclonal antibody 8D7. Results were correlated with cell proliferation characteristics. In flow cytometric analysis, the estrogen receptor (ER)-negative breast cancer cell lines BT-20, MDA-MB-435-S, and SK-BR-3 showed significantly higher PBR expression (relative fluorescence intensity) than the ER-positive cells T47-D, MCF-7 and BT-474 (Pdiazepam-binding inhibitor are possibly involved in the regulation of cell proliferation of human breast cancer cell lines.

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

    Science.gov (United States)

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

    2008-01-01

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

  10. Bacteriophage receptor binding protein based assays for the simultaneous detection of Campylobacter jejuni and Campylobacter coli.

    Science.gov (United States)

    Javed, Muhammad A; Poshtiban, Somayyeh; Arutyunov, Denis; Evoy, Stephane; Szymanski, Christine M

    2013-01-01

    Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barré syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their receptor binding proteins (RBPs), which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host receptor binding protein, Gp047. In the current study, we localized the receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40) and 90% for C. coli (n = 19). CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP). Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.

  11. Bacteriophage receptor binding protein based assays for the simultaneous detection of Campylobacter jejuni and Campylobacter coli.

    Directory of Open Access Journals (Sweden)

    Muhammad A Javed

    Full Text Available Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barré syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their receptor binding proteins (RBPs, which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host receptor binding protein, Gp047. In the current study, we localized the receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40 and 90% for C. coli (n = 19. CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP. Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.

  12. Ritonavir binds to and downregulates estrogen receptors: Molecular mechanism of promoting early atherosclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Jin [Ministry of Education Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Science, Wuhan University, Wuhan 430071 (China); Wang, Ying [Department of Pathophysiology, School of Medicine, Wuhan University, Wuhan 430071 (China); Su, Ke [Department of Nephrology, Renmin Hospital of Wuhan University, Wuhan 430060 (China); Liu, Min [Ministry of Education Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Science, Wuhan University, Wuhan 430071 (China); Hu, Peng-Chao [Department of Pathophysiology, School of Medicine, Wuhan University, Wuhan 430071 (China); Ma, Tian; Li, Jia-Xi [Ministry of Education Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Science, Wuhan University, Wuhan 430071 (China); Wei, Lei [Department of Pathophysiology, School of Medicine, Wuhan University, Wuhan 430071 (China); Zheng, Zhongliang, E-mail: biochem@whu.edu.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Yang, Fang, E-mail: fang-yang@whu.edu.cn [Department of Physiology, School of Medicine, Wuhan University, Wuhan 430071 (China)

    2014-10-01

    Estrogenic actions are closely related to cardiovascular disease. Ritonavir (RTV), a human immunodeficiency virus (HIV) protease inhibitor, induces atherosclerosis in an estrogen-related manner. However, how RTV induce pathological phenotypes through estrogen pathway remains unclear. In this study, we found that RTV increases thickness of coronary artery walls of Sprague Dawley rats and plasma free fatty acids (FFA) levels. In addition, RTV could induce foam cell formation, downregulate both estrogen receptor α (ERα) and ERβ expression, upregulate G protein-coupled estrogen receptor (GPER) expression, and all of them could be partially blocked by 17β-estradiol (E2), suggesting RTV acts as an antagonist for E2. Computational modeling shows a similar interaction with ERα between RTV and 2-aryl indoles, which are highly subtype-selective ligands for ERα. We also found that RTV directly bound to ERα and selectively inhibited the nuclear localization of ERα, and residue Leu536 in the hydrophobic core of ligand binding domain (LBD) was essential for the interaction with RTV. In addition, RTV did not change the secondary structure of ERα-LBD like E2, which explained how ERα lost the capacity of nuclear translocation under the treatment of RTV. All of the evidences suggest that ritonavir acts as an antagonist for 17β-estradiol in regulating α subtype estrogen receptor function and early events of atherosclerosis. - Graphical abstract: RTV directly binds to ERα and Leu536 in the hydrophobic core of ligand binding domain is essential for the interaction. - Highlights: • RTV increases the thickness of rat coronary artery wall and foam cell formation. • RTV downregulates the expression of ERα and ERβ. • RTV inhibits ERα promoter activity. • RTV directly binds to ERα and the key amino acid is Leu536. • RTV inhibits the nuclear translocation of ERα and GPER.

  13. Prenatal exposure to methylmercury alters development of adrenergic receptor binding sites in peripheral sympathetic target tissues

    Energy Technology Data Exchange (ETDEWEB)

    Slotkin, T.A.; Orband, L.; Cowdery, T.; Kavlock, R.J.; Bartolome, J.

    1987-01-01

    In order to assess the impact of prenatal exposure to methylmercury on sympathetic neurotransmission, effects on development of adrenergic receptor binding sites in peripheral tissues was evaluated. In the liver, methylmercury produced a dose-dependent increase in alpha/sub 1/, alpha/sub 2/, and beta-receptor binding of radioliganda throughout the first 5 weeks of postnatal life. Similarly, renal alpha-receptor subtypes showed increased binding capabilities, but binding to alpha-receptor sites was reduced. At least some of the changes in receptors appear to be of functional significance, as physiological reactivity to adrenergic stimulation is altered in the same directions in these two tissues. The actions of methylmercury displayed tissue specificity in that the same receptor populations were largely unaffected in other tissues (lung, heart). These results suggest that methylmercury exposure in utero alters adrenergic responses through targeted effects on postsynaptic receptor populations in specific tissues.

  14. Cycloxaprid insecticide: nicotinic acetylcholine receptor binding site and metabolism.

    Science.gov (United States)

    Shao, Xusheng; Swenson, Tami L; Casida, John E

    2013-08-21

    Cycloxaprid (CYC) is a novel neonicotinoid prepared from the (nitromethylene)imidazole (NMI) analogue of imidacloprid. In this study we consider whether CYC is active per se or only as a proinsecticide for NMI. The IC50 values (nM) for displacing [(3)H]NMI binding are 43-49 for CYC and 2.3-3.2 for NMI in house fly and honeybee head membranes and 302 and 7.2, respectively, in mouse brain membranes, potency relationships interpreted as partial conversion of some CYC to NMI under the assay conditions. The 6-8-fold difference in toxicity of injected CYC and NMI to house flies is consistent with their relative potencies as in vivo nicotinic acetylcholine receptor (nAChR) inhibitors in brain measured with [(3)H]NMI binding assays. CYC metabolism in mice largely involves cytochrome P450 pathways without NMI as a major intermediate. Metabolites of CYC tentatively assigned are five monohydroxy derivatives and one each of dihydroxy, nitroso, and amino modifications. CYC appears be a proinsecticide, serving as a slow-release reservoir for NMI with selective activity for insect versus mammalian nAChRs.

  15. Binding of antibodies to acetylcholine receptors in Electrophorus and Torpedo electroplax membranes

    Science.gov (United States)

    1978-01-01

    Antisera against purified acetylcholine receptors from the electric tissues of Torpedo californica and of Electrophorus electricus were raised in rabbits. The antisera contain antibodies which bind to both autologous and heterologous receptors in solution as shown by an immunoprecipitation assay. Antibodies in both types of antisera bind specifically to the postjunctional membrane on the innervated surface of the intact electroplax from Electrophorus electric tissue as demonstrated by an indirect immunohistochemical procedure using horseradish peroxidase conjugated to anti-rabbit IgG. Only anti- Electrophorus receptor antisera, however, cause inhibition of the receptor-mediated depolarization of the intact Electrophorus electroplax. The lack of inhibition by anti-Torpedo receptor antibodies, which do bind, suggests that the receptor does not undergo extensive movement during activity. The binding of anti-Torpedo antibodies to receptor-rich vesicles prepared by subcellular fractionation of Torpedo electric tissue was demonstrated by both direct and indirect immunohistochemical methods using ferritin conjugates. These vesicles can be conveniently collected and prepared for electron microscopy on Millipore filters, a procedure requiring only 25 micrograms of membrane protein per filter. In addition, it was possible to visualize the binding of anti-Torpedo receptor antibodies directly, without ferritin. These anti-Torpedo receptor antibodies, however, do not inhibit the binding of acetylcholine or of alpha- neurotoxin to receptor in Torpedo microsacs but do inhibit binding of alpha-neurotoxin to Torpedo receptor in Triton X-100 solution. It is likely that the principal antigenic determinants on receptor are at sites other than the acetylcholine-binding sites and that inhibition of receptor function, when it occurs, may be due to a stabilization by antibody binding of an inactive conformational state. PMID:344325

  16. Identification of residues on human receptor DPP4 critical for MERS-CoV binding and entry

    Energy Technology Data Exchange (ETDEWEB)

    Song, Wenfei [Ministry of Education Key Laboratory of Protein Science, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084 (China); Wang, Ying [Comprehensive AIDS Research Center, Research Center for Public Health, School of Medicine, Tsinghua University, Beijing 100084 (China); Wang, Nianshuang; Wang, Dongli [Ministry of Education Key Laboratory of Protein Science, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084 (China); Guo, Jianying; Fu, Lili [Comprehensive AIDS Research Center, Research Center for Public Health, School of Medicine, Tsinghua University, Beijing 100084 (China); Shi, Xuanling, E-mail: shixuanlingsk@tsinghua.edu.cn [Comprehensive AIDS Research Center, Research Center for Public Health, School of Medicine, Tsinghua University, Beijing 100084 (China)

    2014-12-15

    Middle East respiratory syndrome coronavirus (MERS-CoV) infects host cells through binding the receptor binding domain (RBD) on its spike glycoprotein to human receptor dipeptidyl peptidase 4 (hDPP4). Here, we report identification of critical residues on hDPP4 for RBD binding and virus entry through analysis of a panel of hDPP4 mutants. Based on the RBD–hDPP4 crystal structure we reported, the mutated residues were located at the interface between RBD and hDPP4, which potentially changed the polarity, hydrophobic or hydrophilic properties of hDPP4, thereby interfering or disrupting their interaction with RBD. Using surface plasmon resonance (SPR) binding analysis and pseudovirus infection assay, we showed that several residues in hDPP4–RBD binding interface were important on hDPP4–RBD binding and viral entry. These results provide atomic insights into the features of interactions between hDPP4 and MERS-CoV RBD, and also provide potential explanation for cellular and species tropism of MERS-CoV infection. - Highlights: • It has been demonstrated that MERS-CoV infects host cells through binding its envelope spike (S) glycoprotein to the host cellular receptor dipeptidyl peptidase 4 (DPP4). • To identify the critical residues on hDPP4 for RBD binding and virus entry, we constructed a panel of hDPP4 mutants based on structure-guided mutagenesis. • Using surface plasmon resonance (SPR) binding analysis and pseudovirus infection assay, we showed that several residues on hDPP4 had significant impacts on virus/receptor interactions and viral entry. • Our study has provided new insights into the features of interactions between hDPP4 and MERS-CoV RBD, and provides potential explanation for cellular and species tropism of MERS-CoV infection.

  17. A single native ganglioside GM1-binding site is sufficient for cholera toxin to bind to cells and complete the intoxication pathway.

    Science.gov (United States)

    Jobling, Michael G; Yang, Zhijie; Kam, Wendy R; Lencer, Wayne I; Holmes, Randall K

    2012-10-30

    Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM(1) (GM(1)) molecules, and the toxin-GM(1) complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM(1) by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476-1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573-586, 2012), suggesting that multivalent binding to GM(1) is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM(1) binding sites from zero (nonbinding) to five (wild type). We found that a single GM(1) binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM(1) receptors is not essential for toxicity of CT. Through multivalent binding to its lipid receptor, cholera toxin (CT) can remodel cell membranes in ways that may assist host cell invasion. We recently found that CT variants which bind no more than 2 receptor molecules do exhibit toxicity, suggesting that CT may be able to enter cells by coopting an endogenous lipid sorting pathway without clustering receptors. We tested this idea directly by using purified variants

  18. Signal transduction in Dictyostelium fgd A mutants with a defective interaction between surface cAMP receptors and a GTP-binding regulatory protein [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463

    OpenAIRE

    1988-01-01

    Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, including the activation of adenylate or guanylate cyclase and chemotaxis. (b) cAMP induces down- regulation and the covalent modification (presumably phosphorylation) of the cAMP receptor. (c) The inhi...

  19. Presence of insulin receptors in cultured glial C6 cells. Regulation by butyrate.

    Science.gov (United States)

    Montiel, F; Ortiz-Caro, J; Villa, A; Pascual, A; Aranda, A

    1989-01-01

    The presence of insulin receptor and its regulation by butyrate and other short-chain fatty acids was studied in C6 cells, a rat glioma cell line. Intact C6 cells bind 125I-insulin in a rapid, reversible and specific manner. Scatchard analysis of the binding data gives typical curvilinear plots with apparent affinities of approx. 6 nM and 70 nM for the low-affinity (approx. 90% of total) and high-affinity (approx. 10% of total) sites respectively. Incubation with butyrate results in a time- and dose-dependent decrease of insulin binding to C6 cells. A maximal effect was found with 2 mM-butyrate that decreased the receptor by 40-70% after 48 h. Butyrate decreased numbers of receptors of both classes, but did not significantly alter receptor affinity. Other short-chain fatty acids, as well as keto acids, had a similar effect, but with a lower potency. Cycloheximide caused an accumulation of insulin receptors at the cell surface, since insulin binding increased and receptor affinity did not change after incubation with the inhibitor. Simultaneous addition of butyrate and cycloheximide abolished the loss of receptors produced by the fatty acid. In cells preincubated with butyrate, cycloheximide also produced a large increase in receptor numbers, showing that in the absence of new receptor synthesis a large pool of receptors re-appears at the surface of butyrate-treated cells. PMID:2930502

  20. Role of estrogen receptor binding and transcriptional activity in the stimulation of hyperestrogenism and nuclear bodies.

    Science.gov (United States)

    Clark, J H; Hardin, J W; Padykula, H A; Cardasis, C A

    1978-06-01

    The effects of estradiol and nafoxidine on nuclear estrogen receptor binding, RNA polymerase activities, and uterine ultrastructure were studied. Animals were either injected with estradiol, implanted with estradiol/paraffin pellets, or injected with nafoxidine. Animals treated with nafoxidine or estradiol implants showed sustained long-term nuclear retention of estrogen receptor and increased nuclear RNA polymerase activities for up to 72 hr. A single injection of estradiol caused initial increases in these variables which returned to control levels by 24 hr after hormone treatment. Uterine tissue was examined by light and electron microscopy 72 hr after hormone treatments. Uteri from eith estradiol-implanted or nafoxidine-treated animals showed markedly increased hypertrophy of the luminal epithelial cells. Nuclei in sections of the uteri of these hyperestrogenized animals displayed a large number and wide array of nuclear bodies composed of a filamentous capsule and granular cores. We conclude that hyperestrogenization, a condition that eventually results in abnormal cell growth, is correlated with increased and sustained nuclear binding of the estrogen receptor, increased and sustained RNA polymerase activity, and the appearance of nuclear bodies.

  1. Ligand binding affinity and changes in the lateral diffusion of receptor for advanced glycation endproducts (RAGE).

    Science.gov (United States)

    Syed, Aleem; Zhu, Qiaochu; Smith, Emily A

    2016-12-01

    The effect of ligand on the lateral diffusion of receptor for advanced glycation endproducts (RAGE), a receptor involved in numerous pathological conditions, remains unknown. Single particle tracking experiments that use quantum dots specifically bound to hemagglutinin (HA)-tagged RAGE (HA-RAGE) are reported to elucidate the effect of ligand binding on HA-RAGE diffusion in GM07373 cell membranes. The ligand used in these studies is methylglyoxal modified-bovine serum albumin (MGO-BSA) containing advanced glycation end products modifications. The binding affinity between soluble RAGE and MGO-BSA increases by 1.8 to 9.7-fold as the percent primary amine modification increases from 24 to 74% and with increasing negative charge on the MGO-BSA. Ligand incubation affects the HA-RAGE diffusion coefficient, the radius of confinement, and duration of confinement. There is, however, no correlation between MGO-BSA ligand binding affinity with soluble RAGE and the extent of the changes in HA-RAGE lateral diffusion. The ligand induced changes to HA-RAGE lateral diffusion do not occur when cholesterol is depleted from the cell membrane, indicating the mechanism for ligand-induced changes to HA-RAGE diffusion is cholesterol dependent. The results presented here serve as a first step in unraveling how ligand influences RAGE lateral diffusion. Copyright © 2016. Published by Elsevier B.V.

  2. Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor: transfer of binding site to the CXCR3 receptor

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Gerlach, Lars-Ole; Jakobsen, Janus S

    2004-01-01

    AMD3100 is a symmetric bicyclam, prototype non-peptide antagonist of the CXCR4 chemokine receptor. Mutational substitutions at 16 positions located in TM-III, -IV, -V, -VI, and -VII lining the main ligand-binding pocket of the CXCR4 receptor identified three acid residues: Asp(171) (AspIV:20), Asp......, respectively. Metal ion binding in the cyclam rings of AMD3100 increased its dependence on Asp(262) and provided a tighter molecular map of the binding site, where borderline mutational hits became clear hits for the Zn(II)-loaded analog. The proposed binding site for AMD3100 was confirmed by a gradual build...... that AMD3100 binds through interactions with essentially only three acidic anchor-point residues, two of which are located at one end and the third at the opposite end of the main ligand-binding pocket of the CXCR4 receptor. We suggest that non-peptide antagonists with, for example, improved oral...

  3. Modulation of glutamate transport and receptor binding by glutamate receptor antagonists in EAE rat brain.

    Science.gov (United States)

    Sulkowski, Grzegorz; Dąbrowska-Bouta, Beata; Salińska, Elżbieta; Strużyńska, Lidia

    2014-01-01

    The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.

  4. A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells.

    Science.gov (United States)

    Wu, Chunxiao; Wang, Shu

    2012-01-01

    Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.

  5. Differential infection of receptor-modified host cells by receptor-specific influenza viruses.

    Science.gov (United States)

    Carroll, S M; Paulson, J C

    1985-09-01

    Influenza viruses of contrasting receptor specificity have been examined for their ability to infect receptor-modified MDCK cells containing sialyloligosaccharide receptor determinants of defined sequence. Cells were treated with sialidase to remove sialic acid and render them resistant to infection and were then incubated with sialyltransferase and CMP-sialic acid to restore sialic acid in the SA alpha 2,6Gal or SA alpha 2,3Gal linkages. The viruses A/RI/5 + /57 and A/duck/Ukraine/1/63, previously shown to exhibit preferential binding of SA alpha 2,6Gal and SA alpha 2,3Gal linkages, respectively, were found to exhibit differential infection of the receptor-modified cells in accord with their receptor specificity. Coinfection of SA alpha 2,3Gal derivatized cells with a mixture of the two viruses resulted in selective propagation of the SA alpha 2,3Gal-specific A/duck/Ukraine/1/63 virus. The results demonstrate the potential for cell surface receptors to mediate selection of receptor-specific variants of influenza virus.

  6. Quantitation of glucocorticoid receptor DNA-binding dynamics by single-molecule microscopy and FRAP.

    Directory of Open Access Journals (Sweden)

    Femke L Groeneweg

    Full Text Available Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR and the mineralocorticoid receptor (MR, two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (∼ 0.7 s and the other half for longer time periods (∼ 2.3 s. A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (≤ 1 ms interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.

  7. Accelerated Molecular Dynamics Simulations of Ligand Binding to a Muscarinic G-protein Coupled Receptor

    Science.gov (United States)

    Kappel, Kalli; Miao, Yinglong; McCammon, J. Andrew

    2017-01-01

    Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc), and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein-ligand binding, especially the drug recognition of GPCRs. PMID:26537408

  8. Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands.

    Science.gov (United States)

    Chovanec, M; Smetana, K; Purkrábková, T; Holíková, Z; Dvoránková, B; André, S; Pytlík, R; Hozák, P; Plzák, J; Sedo, A; Vacík, J; Gabius, H

    2004-01-01

    The emerging functionality of glycosaminoglycan chains engenders interest in localizing specific binding sites using cytochemical tools. We investigated nuclear binding of labeled heparin, heparan sulfate, a sulfated fucan, chondroitin sulfate, and hyaluronic acid in epidermal keratinocytes, bone marrow stromal cells, 3T3 fibroblasts and glioma cells using chemically prepared biotinylated probes. Binding of the markers was cell-type specific and influenced by extraction of histones, but was not markedly affected by degree of proliferation, differentiation or malignancy. Cell uptake of labeled heparin and other selected probes and their transport into the nucleus also was monitored. Differences between keratinocytes and bone marrow stromal cells were found. Preincubation of permeabilized bone marrow stromal cells with label-free heparin reduced the binding of carrier-immobilized hydrocortisone to its nuclear receptors. Thus, these tools enabled binding sites for glycosaminoglycans to be monitored in routine assays.

  9. Cell and molecular biology of epidermal growth factor receptor.

    Science.gov (United States)

    Ceresa, Brian P; Peterson, Joanne L

    2014-01-01

    The epidermal growth factor receptor (EGFR) has been one of the most intensely studied cell surface receptors due to its well-established roles in developmental biology, tissue homeostasis, and cancer biology. The EGFR has been critical for creating paradigms for numerous aspects of cell biology, such as ligand binding, signal transduction, and membrane trafficking. Despite this history of discovery, there is a continual stream of evidence that only the surface has been scratched. New ways of receptor regulation continue to be identified, each of which is a potential molecular target for manipulating EGFR signaling and the resultant changes in cell and tissue biology. This chapter is an update on EGFR-mediated signaling, and describes some recent developments in the regulation of receptor biology.

  10. The binding of human lipoprotein lipase treated VLDL by the human hepatoma cell line HepG2

    NARCIS (Netherlands)

    Mulder, M.; Wit, E.de; Havekes, L.M.

    1991-01-01

    It has been suggested that besides the LDL-receptor, hepatocytes possess an apo E or remnant receptor. To evaluate which hepatic lipoprotein receptor is involved in VLDL remnant catabolism, we studied the binding of VLDL remnants to HepG2 cells. Native VLDL was obtained from type IIb hyperlipidemic

  11. Ancestral reconstruction of the ligand-binding pocket of Family C G protein-coupled receptors

    OpenAIRE

    Kuang, Donghui; Yao, Yi; MacLean, David; Wang, Minghua; Hampson, David R.; Chang, Belinda S. W.

    2006-01-01

    The metabotropic glutamate receptors (mGluRs) within the Family C subclass of G protein-coupled receptors are crucial modulators of synaptic transmission. However, their closest relatives include a diverse group of sensory receptors whose biological functions are not associated with neurotransmission, raising the question of the evolutionary origin of amino acid-binding Family C receptors. A common feature of most, if not all, functional Family C receptors is the presence of an amino acid-bin...

  12. Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium.

    Science.gov (United States)

    Chinthamani, Sreedevi; Odusanwo, Olutayo; Mondal, Nandini; Nelson, Joel; Neelamegham, Sriram; Baker, Olga J

    2012-04-01

    Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sjögren's syndrome (SS).

  13. Mutation in apolipoprotein B associated with hypobetalipoproteinemia despite decreased binding to the low density lipoprotein receptor

    DEFF Research Database (Denmark)

    Benn, Marianne; Nordestgaard, Børge G; Jensen, Jan Skov;

    2005-01-01

    Mutations in apolipoprotein B (APOB) may reduce binding of low density lipoprotein (LDL) to the LDL receptor and cause hypercholesterolemia. We showed that heterozygotes for a new mutation in APOB have hypobetalipoproteinemia, despite a reduced binding of LDL to the LDL receptor. APOB R3480P hete...

  14. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    Energy Technology Data Exchange (ETDEWEB)

    Bielmann, Regula; Habann, Matthias; Eugster, Marcel R. [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland); Lurz, Rudi [Max-Planck Institute for Molecular Genetics, 14195 Berlin (Germany); Calendar, Richard [Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202 (United States); Klumpp, Jochen, E-mail: jochen.klumpp@hest.ethz.ch [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland); Loessner, Martin J. [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland)

    2015-03-15

    Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.

  15. Structure of human cytomegalovirus UL141 binding to TRAIL-R2 reveals novel, non-canonical death receptor interactions.

    Directory of Open Access Journals (Sweden)

    Ivana Nemčovičová

    2013-03-01

    Full Text Available The TRAIL (TNF-related apoptosis inducing ligand death receptors (DRs of the tumor necrosis factor receptor superfamily (TNFRSF can promote apoptosis and regulate antiviral immunity by maintaining immune homeostasis during infection. In turn, human cytomegalovirus (HCMV expresses immunomodulatory proteins that down-regulate cell surface expression of TNFRSF members as well as poliovirus receptor-related proteins in an effort to inhibit host immune effector pathways that would lead to viral clearance. The UL141 glycoprotein of human cytomegalovirus inhibits host defenses by blocking cell surface expression of TRAIL DRs (by retention in ER and poliovirus receptor CD155, a nectin-like Ig-fold molecule. Here we show that the immunomodulatory function of HCMV UL141 is associated with its ability to bind diverse proteins, while utilizing at least two distinct binding sites to selectively engage TRAIL DRs or CD155. Binding studies revealed high affinity interaction of UL141 with both TRAIL-R2 and CD155 and low affinity binding to TRAIL-R1. We determined the crystal structure of UL141 bound to TRAIL-R2 at 2.1 Å resolution, which revealed that UL141 forms a homodimer that engages two TRAIL-R2 monomers 90° apart to form a heterotetrameric complex. Our structural and biochemical data reveal that UL141 utilizes its Ig-domain to facilitate non-canonical death receptor interactions while UL141 partially mimics the binding site of TRAIL on TRAIL-R2, which we found to be distinct from that of CD155. Moreover, UL141 also binds to an additional surface patch on TRAIL-R2 that is distinct from the TRAIL binding site. Therefore, the breadth of UL141-mediated effects indicates that HCMV has evolved sophisticated strategies to evade the immune system by modulating multiple effector pathways.

  16. Structure of human cytomegalovirus UL141 binding to TRAIL-R2 reveals novel, non-canonical death receptor interactions.

    Science.gov (United States)

    Nemčovičová, Ivana; Benedict, Chris A; Zajonc, Dirk M

    2013-03-01

    The TRAIL (TNF-related apoptosis inducing ligand) death receptors (DRs) of the tumor necrosis factor receptor superfamily (TNFRSF) can promote apoptosis and regulate antiviral immunity by maintaining immune homeostasis during infection. In turn, human cytomegalovirus (HCMV) expresses immunomodulatory proteins that down-regulate cell surface expression of TNFRSF members as well as poliovirus receptor-related proteins in an effort to inhibit host immune effector pathways that would lead to viral clearance. The UL141 glycoprotein of human cytomegalovirus inhibits host defenses by blocking cell surface expression of TRAIL DRs (by retention in ER) and poliovirus receptor CD155, a nectin-like Ig-fold molecule. Here we show that the immunomodulatory function of HCMV UL141 is associated with its ability to bind diverse proteins, while utilizing at least two distinct binding sites to selectively engage TRAIL DRs or CD155. Binding studies revealed high affinity interaction of UL141 with both TRAIL-R2 and CD155 and low affinity binding to TRAIL-R1. We determined the crystal structure of UL141 bound to TRAIL-R2 at 2.1 Å resolution, which revealed that UL141 forms a homodimer that engages two TRAIL-R2 monomers 90° apart to form a heterotetrameric complex. Our structural and biochemical data reveal that UL141 utilizes its Ig-domain to facilitate non-canonical death receptor interactions while UL141 partially mimics the binding site of TRAIL on TRAIL-R2, which we found to be distinct from that of CD155. Moreover, UL141 also binds to an additional surface patch on TRAIL-R2 that is distinct from the TRAIL binding site. Therefore, the breadth of UL141-mediated effects indicates that HCMV has evolved sophisticated strategies to evade the immune system by modulating multiple effector pathways.

  17. Receptor-binding domain of SARS-Cov spike protein: Soluble expression in purification and functional characterization

    Institute of Scientific and Technical Information of China (English)

    Jing Chen; Lin Miao; Jia-Ming Li; Yan-Ying Li; Qing-Yu Zhu; Chang-Lin Zhou; Hong-Qing Fang; Hui-Peng Chen

    2005-01-01

    AIM: To find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability.METHODS: Three fusion tags (glutathione S-transferase,GST; thioredoxin, Trx; maltose-binding protein, MBP),which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli( BL21( DE3 ) and Rosetta-gamiB(DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay.RESULTS: RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2)positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and respectively.CONCLUSION: In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E. coli BL21(DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E. coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

  18. 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.

    2013-01-01

    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. PMID:23255609

  19. 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.

  20. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    Science.gov (United States)

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms.

  1. Probing the GnRH receptor agonist binding site identifies methylated triptorelin as a new anti-proliferative agent

    Directory of Open Access Journals (Sweden)

    Robert P Millar

    2012-06-01

    Full Text Available D-amino acid substitutions at Glycine postion-6 in GnRH-I decapeptide can possess super-agonist activity and enhanced in vivo pharmacokinetics. Agonists elicit growth-inhibition in tumorigenic cells expressing the GnRH receptor above threshold levels. However, new agonists with modified properties are required to improve the anti-proliferative range. Effects of residue substitutions and methylations on tumourigenic HEK293[SCL60] and WPE-1-NB26-3 prostate cells expressing the rat GnRH receptor were compared. Peptides were ranked according to receptor binding affinity, induction of inositol phosphate production and cell growth-inhibition. Analogues possessing D-Trp6 (including Triptorelin, D-Leu6 (including Leuprolide, D-Ala6, D-Lys6, or D-Arg6 exhibited agonist and anti-proliferative activity. Residues His5 or His5,Trp7,Tyr8, corresponding to residues found in GnRH-II , were tolerated, with retention of sub-nanomolar/low nanomolar binding affinities and EC50s for receptor activation and IC50s for cell growth-inhibition. His5D-Arg6-GnRH-I exhibited reduced binding affinity and potency, effective in the mid-nanomolar range. However, all GnRH-II-like analogues were less potent than Triptorelin. By comparison, three methylated-Trp6 Triptorelin variants showed differential binding, receptor activation and anti-proliferation potency. Significantly, 5-Methyl-DL-Trp6-Triptorelin was equipotent to triptorelin. Subsequent studies should determine whether pharmacologically enhanced derivatives of Triptorelin can be developed by further alkylations, without substitutions or cleavable cytotoxic adducts, to improve the extent of growth-inhibition of tumour cells expressing the GnRH receptor.

  2. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M;

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression...... of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell...

  3. The receptor binding fragment of alpha-fetoprotein is a promising new vector for the selective delivery of antineoplastic agents.

    Science.gov (United States)

    Posypanova, Galina A; Makarov, Vladimir A; Savvateeva, Mariya V; Bereznikova, Anastasiya V; Severin, Evgeny S

    2013-05-01

    The alpha-fetoprotein (AFP) binding protein, a putative AFP receptor, is a tumour marker that is present on the surfaces of malignant cells. AFP enters cells through receptor-mediated endocytosis. The recombinant C-terminal fragment of AFP (AFP-3BC, which consists of amino acid residues 473-596) was obtained by the expression in Escherichia coli. AFP-3BC was shown to be bound specifically to the AFP putative receptor on tumour cells and accumulated by endocytosis in these cells in a similar manner to that of full-length human AFP. In lymphocytes, the binding and endocytosis of AFP-3BC were absent. Thus, the AFP receptor binding site was shown experimentally to be located within the AFP-3BC sequence. A conjugate of synthesised AFP-3BC with the antitumour antibiotic doxorubicin (DOX-AFP-3BC) demonstrated high antitumour activity in vitro. Thus, AFP-3BC can be used successfully as a vector for the targeted selective delivery of drugs into tumour cells.

  4. Reassessment of the unique mode of binding between angiotensin II type 1 receptor and their blockers.

    Directory of Open Access Journals (Sweden)

    Shin-Ichiro Miura

    Full Text Available While the molecular structures of angiotensin II (Ang II type 1 (AT1 receptor blockers (ARBs are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT1 receptor, different positions of the AT1 receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT1 receptor, Tyr(113, Tyr(184, Lys(199, His(256 and Gln(257 using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT1 receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr(113 in the AT1 receptor and the hydroxyl group of olmesartan, between Lys(199 and carboxyl or tetrazole groups, and between His(256 or Gln(257 and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys(199, His(256 and Gln(257 of AT1 receptor. Lys(199 in the AT1 receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys(199. The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr(113. On the other hand, the n-butyl group of irbesartan may bind to Tyr(113. In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT1 receptor and for the formation of unique modes of binding.

  5. Lipid raft-dependent FcepsilonRI ubiquitination regulates receptor endocytosis through the action of ubiquitin binding adaptors.

    Directory of Open Access Journals (Sweden)

    Rosa Molfetta

    Full Text Available The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcepsilonRI expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcepsilonRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcepsilonRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.

  6. Relationship between internalization and calcitonin-induced receptor loss in T 47D cells

    Energy Technology Data Exchange (ETDEWEB)

    Findlay, D.M.; Martin, T.J.

    1984-07-01

    Exposure of T 47D human breast cancer cells to salmon calcitonin (sCT) resulted in a reduction of binding capacity for (/sup 125/I)iodo-sCT in washed cells. The reduction was both time and concentration dependent. Recovery of binding capacity in CT-pretreated T 47D cells occurred in the absence of CT, but was prevented by inhibitors of protein synthesis. Studies were carried out to determine the mechanism of CT-induced reduction of binding capacity. It was found that at 37/sup 0/ C sCT induced a time-dependent loss of cell surface receptors, so that initially the lost binding capacity was largely reclaimable by acid treatment, whereas after longer exposure to sCT, acid treatment was much less effective in regenerating binding capacity. The CT-induced reduction in binding capacity was not observed when cells were pretreated with sCT at 4 C or in the presence of inhibitors of cellular metabolic energy. These results are consistent with the view that initially CT-induced loss of CT receptors in T 47D cells is primarily due to occupancy of cell-surface receptors and later to a reduction in the concentration of cell-surface receptors mediated by an energy requiring internalization process involving the CT-receptor complex; reappearance of receptors requires new protein synthesis.

  7. Rat neuronal nicotinic acetylcholine receptors containing a7 subunit: pharmacological properties of ligand binding and function

    Institute of Scientific and Technical Information of China (English)

    Yingxian XIAO; Galya R ABDRAKHMANOVA; Maryna BAYDYUK; Susan HERNANDEZ; Kenneth J KELLAR

    2009-01-01

    Aim: To compare pharmacological properties of heterologously expressed homomeric a7 nicotinic acetylcholine receptors (a.7 nAChRs) with those of native nAChRs containing a.7 subunit (a.7* nAChRs) in rat hippocampus and cerebral cortex. Methods: We established a stably transfected HEK-293 cell line that expresses homomeric rat a7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat a.7* nAChRs in rat hippocampus and cerebral cortex. We used [125IJ-a-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the a.7 nAChRs expressed in this cell line were studied using whole-cell current recording.Results: The newly established cell line, KXa7Rl, expresses homomeric a7 nAChRs that bind [125I]-a-bungarotoxin with a Kd value of 0.38±0.06 nmol/L, similar to Kj values of native rat a.7* nAChRs from hippocampus (Kd=0.28±0.03 nmol/L) and cerebral cortex (Kd=0.33±0.05 nmol/L). Using whole-cell current recording, the homomeric a7 nAChRs expressed in the cells were activated by acetylcholine and (-)-nicotine with EC50 values of 280±19 nmol/L and 180±40 nmol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of a.7 nAChRs, a-bungarotoxin (IC5o=19±2 nmol/L) and methyllycaconitine (IC50=100±10 pmol/L). A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric a.7 nAChRs and native a.7* receptors in rat brain, but it also revealed several notable differences.Conclusion: This newly established stable cell line should be very useful for studying the properties of homomeric a7 nAChRs and comparing these properties to native a.7* nAChRs.

  8. Riboswitches as hormone receptors: hypothetical cytokinin-binding riboswitches in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Downes Brian

    2010-10-01

    Full Text Available Abstract Background Riboswitches are mRNA elements that change conformation when bound to small molecules. They are known to be key regulators of biosynthetic pathways in both prokaryotes and eukaryotes. Presentation of the Hypothesis The hypothesis presented here is that riboswitches function as receptors in hormone perception. We propose that riboswitches initiate or integrate signaling cascades upon binding to classic signaling molecules. The molecular interactions for ligand binding and gene expression control would be the same as for biosynthetic pathways, but the context and the cadre of ligands to consider is dramatically different. The hypothesis arose from the observation that a compound used to identify adenine binding RNA sequences is chemically similar to the classic plant hormone, or growth regulator, cytokinin. A general tenet of the hypothesis is that riboswitch-binding metabolites can be used to make predictions about chemically related signaling molecules. In fact, all cell permeable signaling compounds can be considered as potential riboswitch ligands. The hypothesis is plausible, as demonstrated by a cursory review of the transcriptome and genome of the model plant Arabidopsis thaliana for transcripts that i contain an adenine aptamer motif, and ii are also predicted to be cytokinin-regulated. Here, one gene, CRK10 (for Cysteine-rich Receptor-like Kinase 10, At4g23180, contains an adenine aptamer-related sequence and is down-regulated by cytokinin approximately three-fold in public gene expression data. To illustrate the hypothesis, implications of cytokinin-binding to the CRK10 mRNA are discussed. Testing the hypothesis At the broadest level, screening various cell permeable signaling molecules against random RNA libraries and comparing hits to sequence and gene expression data bases could determine how broadly the hypothesis applies. Specific cases, such as CRK10 presented here, will require experimental validation of direct

  9. Structures and receptor binding of hemagglutinins from human-infecting H7N9 influenza viruses.

    Science.gov (United States)

    Shi, Yi; Zhang, Wei; Wang, Fei; Qi, Jianxun; Wu, Ying; Song, Hao; Gao, Feng; Bi, Yuhai; Zhang, Yanfang; Fan, Zheng; Qin, Chengfeng; Sun, Honglei; Liu, Jinhua; Haywood, Joel; Liu, Wenjun; Gong, Weimin; Wang, Dayan; Shu, Yuelong; Wang, Yu; Yan, Jinghua; Gao, George F

    2013-10-11

    An avian-origin human-infecting influenza (H7N9) virus was recently identified in China. We have evaluated the viral hemagglutinin (HA) receptor-binding properties of two human H7N9 isolates, A/Shanghai/1/2013 (SH-H7N9) (containing the avian-signature residue Gln(226)) and A/Anhui/1/2013 (AH-H7N9) (containing the mammalian-signature residue Leu(226)). We found that SH-H7N9 HA preferentially binds the avian receptor analog, whereas AH-H7N9 HA binds both avian and human receptor analogs. Furthermore, an AH-H7N9 mutant HA (Leu(226) → Gln) was found to exhibit dual receptor-binding property, indicating that other amino acid substitutions contribute to the receptor-binding switch. The structures of SH-H7N9 HA, AH-H7N9 HA, and its mutant in complex with either avian or human receptor analogs show how AH-H7N9 can bind human receptors while still retaining the avian receptor-binding property.

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

    Science.gov (United States)

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

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  11. Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

    Institute of Scientific and Technical Information of China (English)

    葛猛; 徐俊杰; 李冰; 董大勇; 宋小红; 郭强; 赵剑; 陈薇

    2004-01-01

    The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E. coli. Signal sequence of the outer membrane protein A (OmpA) of E. coli was attached to the 5' end of the gene encoding protective antigen receptor-binding domain (the 4th domain of PA, PALM). The plasmid carrying the fusion gene was then transformed into E. coli and induced to express recombinant PAlM by IFFG. The recombinant protein was purified by chromatography and then identified by N-terrainal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ionexchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E. coli. The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.

  12. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    Science.gov (United States)

    Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

    2017-01-01

    Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

  13. Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid cells

    Energy Technology Data Exchange (ETDEWEB)

    Tseng, Y.L.; Burman, K.D.; Lahiri, S.; Abdelrahim, M.M.; D' Avis, J.C.; Wartofsky, L. (Walter Reed Army Medical Center, Washington, DC (USA))

    1991-03-01

    In a prior study of atrial natriuretic peptide (ANP) binding to cultured thyroid cells, we reported that at 4 C, more than 95% of bound ANP is recovered on cell membranes, with negligible ANP internalization observed. Since ANP binding was inhibited by TSH, we have further studied TSH effects on postbinding ANP processing to determine whether this phenomenon reflects enhanced endocytosis of the ANP-receptor complex. An ANP chase study was initiated by binding (125I) ANP to thyroid cells at 4 C for 2 h, followed by incubation at 37 C. ANP processing was then traced by following 125I activity at various time intervals in three fractions: cell surface membranes, incubation medium, and inside the cells. Radioactivity released into medium represented processed ANP rather than ANP dissociated from surface membranes, since prebound (125I)ANP could not be competitively dissociated by a high concentration of ANP (1 mumol/L) at 37 C. Chase study results showed that prebound ANP quickly disappeared from cell membranes down to 34% by 30 min. Internalized ANP peaked at 10 min, with 21% of initial prebound ANP found inside the cells. At the same time, radioactivity recovered in incubation medium sharply increased between 10-30 min from 8% to 52%. Preincubation of cells with chloroquine (which blocks degradation of the ANP-receptor complex by inhibiting lysosomal hydrolase) caused a 146% increase in internalized (125I)ANP by 30 min (39% compared to 15% control), while medium radioactivity decreased from 52% to 16%, suggesting that processing of the receptor complex is mediated via lysosomal enzymes. In chase studies employing cells pretreated with chloroquine, TSH stimulated the internalization rate of ANP-receptor complex. By 30 min, TSH significantly reduced the membrane-bound ANP, and the decrease was inversely correlated to the increase in internalized radioactivity.

  14. A robust homogeneous binding assay for α4β2 nicotinic acetylcholine receptor

    Institute of Scientific and Technical Information of China (English)

    Xin HUI; Jie GAO; Xin XIE; Naoki SUTO; Tsuyoshi OGIKU; Ming-Wei WANG

    2005-01-01

    Aim: To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel α4β2 nicotinic acetylcholine receptor (nAChR) modulators. Methods: Membrane preparation of HEK293 cells expressing α4β2 nAChR, [3H]cytisine and wheat germ agglutinin (WGA)-coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32 000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. Results: IC50 values of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported elsewhere. A total of 54 compounds, showing more than 60% competitive inhibition on [3H]cytisine binding to α4β2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to α4β2 nAChR (Ki<2 μmol/L). Eight compounds displayed antagonistic effects with >50% inhibition on ABT-594-induced calcium mobilization while none showed any agonist activity. Conclusions: This homogeneous binding assay is a highly efficient,amenable to automation and robust tool to screen potential α4β2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.

  15. Cholesterol efflux via ATP-binding cassette transporter A1 (ABCA1) and cholesterol uptake via the LDL receptor influences cholesterol-induced impairment of beta cell function in mice

    NARCIS (Netherlands)

    Kruit, J. K.; Kremer, P. H. C.; Dai, L.; Tang, R.; Ruddle, P.; de Haan, W.; Brunham, L. R.; Verchere, C. B.; Hayden, M. R.

    2010-01-01

    Cellular cholesterol accumulation is an emerging mechanism for beta cell dysfunction in type 2 diabetes. Absence of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) results in increased islet cholesterol and impaired insulin secretion, indicating that impaired cholesterol effl

  16. Pairwise comparison of {sup 89}Zr- and {sup 124}I-labeled cG250 based on positron emission tomography imaging and nonlinear immunokinetic modeling: in vivo carbonic anhydrase IX receptor binding and internalization in mouse xenografts of clear-cell renal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Cheal, Sarah M.; Punzalan, Blesida; Doran, Michael G.; Osborne, Joseph R. [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York, NY (United States); Evans, Michael J. [Memorial Sloan-Kettering Cancer Center, Human Oncology and Pathogenesis Program, New York, NY (United States); Lewis, Jason S. [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Program in Molecular Pharmacology and Chemistry, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Radiochemistry and Imaging Sciences Service, New York, NY (United States); Zanzonico, Pat [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Molecular Pharmacology and Therapy Service, New York, NY (United States); Memorial-Sloan Kettering Cancer Center, New York, NY (United States); Larson, Steven M. [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Program in Molecular Pharmacology and Chemistry, New York, NY (United States); Memorial Sloan-Kettering Cancer Center, Molecular Pharmacology and Therapy Service, New York, NY (United States)

    2014-05-15

    The PET tracer, {sup 124}I-cG250, directed against carbonic anhydrase IX (CAIX) shows promise for presurgical diagnosis of clear-cell renal cell carcinoma (ccRCC) (Divgi et al. in Lancet Oncol 8:304-310, 2007; Divgi et al. in J Clin Oncol 31:187-194, 2013). The radiometal {sup 89}Zr, however, may offer advantages as a surrogate PET nuclide over {sup 124}I in terms of greater tumor uptake and retention (Rice et al. in Semin Nucl Med 41:265-282, 2011). We have developed a nonlinear immunokinetic model to facilitate a quantitative comparison of absolute uptake and antibody turnover between {sup 124}I-cG250 and {sup 89}Zr-cG250 using a human ccRCC xenograft tumor model in mice. We believe that this unique model better relates quantitative imaging data to the salient biological features of tumor antibody-antigen binding and turnover. We conducted experiments with {sup 89}Zr-cG250 and {sup 124}I-cG250 using a human ccRCC cell line (SK-RC-38) to characterize the binding affinity and internalization kinetics of the two tracers in vitro. Serial PET imaging was performed in mice bearing subcutaneous ccRCC tumors to simultaneously detect and quantify time-dependent tumor uptake in vivo. Using the known specific activities of the two tracers, the equilibrium rates of antibody internalization and turnover in the tumors were derived from the PET images using nonlinear compartmental modeling. The two tracers demonstrated virtually identical tumor cell binding and internalization but showed markedly different retentions in vitro. Superior PET images were obtained using {sup 89}Zr-cG250, owing to the more prolonged trapping of the radiolabel in the tumor and simultaneous washout from normal tissues. Estimates of cG250/CAIX complex turnover were 1.35 - 5.51 x 10{sup 12} molecules per hour per gram of tumor (20 % of receptors internalized per hour), and the ratio of {sup 124}I/{sup 89}Zr atoms released per unit time by tumor was 17.5. Pairwise evaluation of {sup 89}Zr-cG250 and {sup

  17. N-terminal truncation enables crystallization of the receptor-binding domain of the FedF bacterial adhesin

    Energy Technology Data Exchange (ETDEWEB)

    De Kerpel, Maia; Van Molle, Inge [Department of Ultrastructure, Vrije Universiteit Brussel (VUB), Flanders Interuniversity Institute for Biotechnology (VIB), Pleinlaan 2, 1050 Brussels (Belgium); Brys, Lea [Department of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Flanders Interuniversity Institute for Biotechnology (VIB), Pleinlaan 2, 1050 Brussels (Belgium); Wyns, Lode; De Greve, Henri; Bouckaert, Julie, E-mail: bouckaej@vub.ac.be [Department of Ultrastructure, Vrije Universiteit Brussel (VUB), Flanders Interuniversity Institute for Biotechnology (VIB), Pleinlaan 2, 1050 Brussels (Belgium)

    2006-12-01

    The N-terminal receptor-binding domain of the FedF adhesin from enterotoxigenic E. coli has been crystallized. This required the deletion of its first 14 residues, which are also cleaved off naturally. FedF is the two-domain tip adhesin of F18 fimbriae from enterotoxigenic Escherichia coli. Bacterial adherence, mediated by the N-terminal receptor-binding domain of FedF to carbohydrate receptors on intestinal microvilli, causes diarrhoea and oedema disease in newly weaned piglets and induces the secretion of Shiga toxins. A truncate containing only the receptor-binding domain of FedF was found to be further cleaved at its N-terminus. Reconstruction of this N-terminal truncate rendered FedF amenable to crystallization, resulting in crystals with space group P2{sub 1}2{sub 1}2{sub 1} and unit-cell parameters a = 36.20, b = 74.64, c = 99.03 Å that diffracted to beyond 2 Å resolution. The binding specificity of FedF was screened for on a glycan array, exposing 264 glycoconjugates, to identify specific receptors for cocrystallization with FedF.

  18. Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

    OpenAIRE

    Diao, Jingyu; Pantua, Homer; Ngu, Hai; Komuves, Laszlo; Diehl, Lauri; Schaefer, Gabriele; Kapadia, Sharookh B.

    2012-01-01

    While epidermal growth factor receptor (EGFR) has been shown to be important in the entry process for multiple viruses, including hepatitis C virus (HCV), the molecular mechanisms by which EGFR facilitates HCV entry are not well understood. Using the infectious cell culture HCV model (HCVcc), we demonstrate that the binding of HCVcc particles to human hepatocyte cells induces EGFR activation that is dependent on interactions between HCV and CD81 but not claudin 1. EGFR activation can also be ...

  19. Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

    Science.gov (United States)

    Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A; Manna, Sunil K

    2010-02-19

    The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

  20. Comparison of the Binding and Functional Properties of Two Structurally Different D2 Dopamine Receptor Subtype Selective Compounds

    Science.gov (United States)

    2012-01-01

    We previously reported on the synthesis of substituted phenyl-4-hydroxy-1-piperidyl indole analogues with nanomolar affinity at D2 dopamine receptors, ranging from 10- to 100-fold selective for D2 compared to the D3 dopamine receptor subtype. More recently, we evaluated a panel of aripiprazole analogues, identifying several analogues that also exhibit D2 vs D3 dopamine receptor binding selectivity. These studies further characterize the intrinsic efficacy of the compound with the greatest binding selectivity from each chemical class, 1-((5-methoxy-1H-indol-3-yl)methyl)-4-(4-(methylthio)phenyl)piperidin-4-ol (SV 293) and 7-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (SV-III-130s), using an adenylyl cyclase inhibition assay, a G-protein-coupled inward-rectifying potassium (GIRK) channel activation assay, and a cell based phospho-MAPK (pERK1/2) assay. SV 293 was found to be a neutral antagonist at D2 dopamine receptors using all three assays. SV-III-130s is a partial agonist using an adenylyl cyclase inhibition assay but an antagonist in the GIRK and phospho ERK1/2 assays. To define the molecular basis for the binding selectivity, the affinity of these two compounds was evaluated using (a) wild type human D2 and D3 receptors and (b) a panel of chimeric D2/D3 dopamine receptors. Computer-assisted modeling techniques were used to dock these compounds to the human D2 and D3 dopamine receptor subtypes. It is hoped that these studies on D2 receptor selective ligands will be useful in the future design of (a) receptor selective ligands used to define the function of D2-like receptor subtypes, (b) novel pharmacotherapeutic agents, and/or (c) in vitro and in vivo imaging agents. PMID:23259040

  1. Research Resource: Androgen Receptor Activity Is Regulated Through the Mobilization of Cell Surface Receptor Networks.

    Science.gov (United States)

    Hsiao, Jordy J; Ng, Brandon H; Smits, Melinda M; Martinez, Harryl D; Jasavala, Rohini J; Hinkson, Izumi V; Fermin, Damian; Eng, Jimmy K; Nesvizhskii, Alexey I; Wright, Michael E

    2015-08-01

    The aberrant expression of androgen receptor (AR)-dependent transcriptional programs is a defining pathology of the development and progression of prostate cancers. Transcriptional cofactors that bind AR are critical determinants of prostate tumorigenesis. To gain a deeper understanding of the proteins linked to AR-dependent gene transcription, we performed a DNA-affinity chromatography-based proteomic screen designed to identify proteins involved in AR-mediated gene transcription in prostate tumor cells. Functional experiments validated the coregulator roles of known AR-binding proteins in AR-mediated transcription in prostate tumor cells. More importantly, novel coregulatory functions were detected in components of well-established cell surface receptor-dependent signal transduction pathways. Further experimentation demonstrated that components of the TNF, TGF-β, IL receptor, and epidermal growth factor signaling pathways modulated AR-dependent gene transcription and androgen-dependent proliferation in prostate tumor cells. Collectively, our proteomic dataset demonstrates that the cell surface receptor- and AR-dependent pathways are highly integrated, and provides a molecular framework for understanding how disparate signal-transduction pathways can influence AR-dependent transcriptional programs linked to the development and progression of human prostate cancers.

  2. Recognition of coxiella burnetii by toll-like receptors and nucleotide-binding oligomerization domain-like receptors

    NARCIS (Netherlands)

    Ammerdorffer, Anne; Schoffelen, Teske; Gresnigt, Mark S.; Oosting, Marije; Brok, Den Martijn H.; Abdollahi-Roodsaz, Shahla; Kanneganti, Thirumala Devi; Jong, De Dirk J.; Deuren, Van Marcel; Roest, Hendrik Jan; Rebel, Johanna M.J.; Netea, Mihai G.; Joosten, Leo A.B.; Sprong, Tom

    2015-01-01

    Background. Infection with Coxiella burnetii can lead to acute and chronic Q fever. Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR6, nucleotide-binding oligomerization domain receptor 1 (NOD1), NOD2, and the mitogen-activated protein kinases are central in the innate immune response against

  3. Recognition of Coxiella burnetii by Toll-like Receptors and Nucleotide-Binding Oligomerization Domain-like Receptors

    NARCIS (Netherlands)

    Ammerdorffer, A.; Schoffelen, T.; Gresnigt, M.S.; Oosting, M.; Brok, M.H.M.G.M. den; Abdollahi-Roodsaz, S.; Kanneganti, T.D.; Jong, D.J. de; Deuren, M. van; Roest, H.J.; Rebel, J.M.; Netea, M.G.; Joosten, L.A.B.; Sprong, T.

    2015-01-01

    BACKGROUND: Infection with Coxiella burnetii can lead to acute and chronic Q fever. Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR6, nucleotide-binding oligomerization domain receptor 1 (NOD1), NOD2, and the mitogen-activated protein kinases are central in the innate immune response against

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    Recent advances in structural biology for G-protein-coupled receptors (GPCRs) have provided new opportunities to improve the definition of the transmembrane binding pocket. Here a reference set of 44 residue positions accessible for ligand binding was defined through detailed analysis of all curr...

  5. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

    Science.gov (United States)

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique

    2011-01-01

    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

  6. Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization.

    Science.gov (United States)

    Sánchez-Vallet, Andrea; Saleem-Batcha, Raspudin; Kombrink, Anja; Hansen, Guido; Valkenburg, Dirk-Jan; Thomma, Bart P H J; Mesters, Jeroen R

    2013-07-02

    While host immune receptors detect pathogen-associated molecular patterns to activate immunity, pathogens attempt to deregulate host immunity through secreted effectors. Fungi employ LysM effectors to prevent recognition of cell wall-derived chitin by host immune receptors, although the mechanism to compete for chitin binding remained unclear. Structural analysis of the LysM effector Ecp6 of the fungal tomato pathogen Cladosporium fulvum reveals a novel mechanism for chitin binding, mediated by intrachain LysM dimerization, leading to a chitin-binding groove that is deeply buried in the effector protein. This composite binding site involves two of the three LysMs of Ecp6 and mediates chitin binding with ultra-high (pM) affinity. Intriguingly, the remaining singular LysM domain of Ecp6 binds chitin with low micromolar affinity but can nevertheless still perturb chitin-triggered immunity. Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex. DOI:http://dx.doi.org/10.7554/eLife.00790.001.

  7. Involvement of Activating NK Cell Receptors and Their Modulation in Pathogen Immunity

    Directory of Open Access Journals (Sweden)

    Francesco Marras

    2011-01-01

    Full Text Available Natural Killer (NK cells are endowed with cell-structure-sensing receptors providing inhibitory protection from self-destruction (inhibitory NK receptors, iNKRs, including killer inhibitory receptors and other molecules and rapid triggering potential leading to functional cell activation by Toll-like receptors (TLRs, cytokine receptors, and activating NK cell receptors including natural cytotoxicity receptors (NCRs, i.e., NKp46, NKp46, and NKp44. NCR and NKG2D recognize ligands on infected cells which may be endogenous or may directly bind to some structures derived from invading pathogens. In this paper, we address the known direct or indirect interactions between activating receptors and pathogens and their expression during chronic HIV and HCV infections.

  8. The blunt trichoid sensillum of female mosquitoes, Anopheles gambiae: odorant binding protein and receptor types.

    Science.gov (United States)

    Schultze, Anna; Breer, Heinz; Krieger, Jürgen

    2014-01-01

    In order to find a blood host and to select appropriate oviposition sites female Anopheles gambiae mosquitoes rely on olfactory cues which are sensed by olfactory sensory neurons (OSNs) located within morphologically different sensilla hairs. While the sharp type trichoid sensilla are most abundant and intensely studied, the striking blunt type trichoid sensilla exist only in small numbers and their specific function is unknown. It has been suggested that they may play a role in the detection of chemical cues indicating oviposition sites. With the aim of identifying molecular elements in blunt type trichoid sensilla, which may be relevant for chemosensory function of this sensillum type, experiments were performed which include whole mount fluorescence in situ hybridization (WM-FISH) and fluorescence immunohistochemistry (WM-FIHC). The studies were concentrated on odorant binding proteins (AgOBPs) and odorant receptors (AgORs). WM-FISH approaches using a probe for the plus-C class AgOBP47 led to the labeling of cells, which resembled in number and antennal distribution pattern the blunt type trichoid sensilla. Moreover, WM-FIHC with an antiserum for AgOBP47 allowed to assign the AgOBP47-expressing cells to blunt type trichoid sensilla and to allocate the protein within the sensillum hair shafts. The result of double WM-FISH-experiments and combined WM-FIHC/FISH approaches indicated that the AgOBP47-expressing cells are co-localized with cells, which express AgOR11, AgOR13 and AgOR55. In addition, it turned out that the two receptor types AgOR13 and AgOR55 are co-expressed in the same cells. Together, the results indicate that the blunt type trichoid sensilla contain a characteristic binding protein, plus-C AgOBP47, in the sensillum lymph and two sensory neurons, one cell which express the odorant receptor AgOR11 and a second cell which express the receptor types AgOR13 and AgOR55. The expression of characteristic chemosensory elements in blunt type trichoid

  9. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein.

    OpenAIRE

    Gray, P W; Barrett, K; Chantry, D; Turner, M.; Feldmann, M

    1990-01-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNF alpha with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surf...

  10. Effects of vitamin B-6 nutrition on benzodiazepine (BDZ) receptor binding in the developing rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Borek, J.P.; Guilarte, T.R. (Johns Hopkins Univ., Baltimore, MD (United States))

    1990-02-26

    A dietary deficiency of vitamin B-6 promotes seizure activity in neonatal animals and human infants. Previous studied have shown that neonatal vitamin B-6 deprivation results in reduced levels of brain gamma-aminobutyric acid (GABA) and increased binding at the GABA site of the GABA/BDZ receptor complex. Since the GABA and BDZ receptors are allosterically linked, this study was undertaken to determine if vitamin B-6 deprivation had an effect on BDZ receptor binding. Benzodiazepine receptor binding isotherms using {sup 3}H-flunitrazepam as ligand were performed in the presence and absence of 10 {mu}M GABA. The results indicate a significant increase in the binding affinity (Kd) in the presence of GABA in cerebellar membranes from deficient rat pups at 14 days of age with no effect on receptor number (Bmax). By 28 days of age, the increase in Kd was no longer present. No change in Kd or Bmax was observed in cortical tissue from deficient animals at 14 or 28 days of age. Preliminary studies of GABA-enhancement of {sup 3}H-flunitrazepam binding indicate that vitamin B-6 deficiency also induces alterations in the ability of GABA to enhance BZD receptor binding. In summary, these results indicate that the effects of vitamin B-6 deprivation on BDZ receptor binding are region specific and age related.

  11. Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists

    Energy Technology Data Exchange (ETDEWEB)

    Fryer, A.D.; El-Fakahany, E.E. (Univ. of Maryland, Baltimore (USA))

    1990-01-01

    Heterogeneity of the muscarinic receptor population in the rat central and peripheral lung was found in competition binding experiments against ({sup 3}H)quinuclidinyl benzilate (({sup 3}H)QNB) using the selective antagonists pirenzepine, AF-DX 116 and hexahydrosiladifenidol (HHSiD). Pirenzepine displaced ({sup 3}H)QNB with low affinity from preparations of central airways indicating the absence of M{sub 1} receptors in the trachea and bronchi. Muscarinic receptors in the central airways are comprised of both M{sub 2} and M{sub 3} receptors since AF-DX 116, an M{sub 2}-selective antagonist, bound with high affinity to 70% of the available sites while HHSiD, an M{sub 3}-selective antagonist bound with high affinity to the remaining binding sites. In the peripheral lung, pirenzepine bound with high affinity to 14% of the receptor population, AF-DX 116 bound with high affinity 79% of the binding sites while HHSiD bound with high affinity to 18% of the binding sites. The presence of M{sub 1} receptors in the peripheral airways but not in the central airways was confirmed using ({sup 3}H)telenzepine, an M{sub 1} receptor ligand. ({sup 3}H)Telenzepine showed specific saturable binding to 8% of ({sup 3}H)QNB labeled binding sites in homogenates of rat peripheral lung, while there was no detectable specific binding in homogenates of rat trachea or heart.

  12. The unsialylated subpopulation of recombinant activated factor VII binds to the asialo-glycoprotein receptor (ASGPR) on primary rat hepatocytes.

    Science.gov (United States)

    Seested, Torben; Nielsen, Hanne M; Christensen, Erik I; Appa, Rupa S

    2010-12-01

    Recombinant activated factor VII (rFVIIa; NovoSeven®) is a heterogeneously glycosylated serine protease used for treatment of haemophiliacs with inhibitors. The drug substance contains a subpopulation consisting of ~20% of rFVIIa molecules which are unsialylated and consists of carbohydrate moieties with terminally exposed galactose and N-acetyl-D-galactosamine (GalNAc). Recently, data from an in situ perfused liver model showed that a subpopulation of rFVIIa, appearing to be unsialylated rFVIIa, was cleared by the liver, thus suggesting a carbohydrate-moiety mediated mechanism. The parenchymal cells of the liver, hepatocytes, are known to abundantly express functional carbohydrate-specific receptors and in this study we therefore used primary rat hepatocytes to study binding and intracellular fate of rFVIIa at a cellular level. Immunofluorescence microscopy showed that rFVIIa was distributed into distinct intracellular vesicles and electron microscopic autoradiography revealed that radioiodinated rFVIIa distributed only into cytoplasmic free vesicles resembling endosomes and lysosomes. These findings suggest that endocytosis of rFVIIa in hepatocytes could be partly mediated via initial membrane binding to a receptor. Quantitative binding studies showed that the presence of excess unlabelled asialo-orosomucoid, asialo-rFVIIa and GalNAc significantly decreased binding of 125I-rFVIIa. An antibody which specifically binds to the carbohydrate recognition domain of the asialoglycoprotein receptor (ASGPR) significantly decreased binding of asialo-rFVIIa by ~36% and rFVIIa by ~19%. Together our data showed that a receptor-mediated mechanism involving the ASGPR is able to bind a subpopulation of unsialylated rFVIIa, while a hepatic mechanism for binding and clearing sialylated rFVIIa is still unknown.

  13. Evolutionary vignettes of natural killer cell receptors.

    Science.gov (United States)

    Sambrook, Jennifer G; Beck, Stephan

    2007-10-01

    The discovery of novel immune receptors has led to a recent renaissance of research into the innate immune system, following decades of intense research of the adaptive immune system. Of particular interest has been the discovery of the natural killer (NK) cell receptors which, depending on type, interact with classical or non-classical MHC class I antigens of the adaptive immune system, thus functioning at the interface of innate and adaptive immunity. Here, we review recent progress with respect to two such families of NK receptors, the killer immunoglobulin-like receptors (KIRs) and the killer cell lectin-like receptors (KLRs), and attempt to trace their evolution across vertebrates.

  14. Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

    Energy Technology Data Exchange (ETDEWEB)

    Lee, J.H.; el-Fakahany, E.E.

    1985-06-01

    The binding properties of (-)-(/sup 3/H)quinuclidinyl benzilate and (/sup 3/H) N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-(/sup 3/H)quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of (/sup 3/H)N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when (/sup 3/H)N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-(/sup 3/H) quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.

  15. CD44 is the principal cell surface receptor for hyaluronate.

    Science.gov (United States)

    Aruffo, A; Stamenkovic, I; Melnick, M; Underhill, C B; Seed, B

    1990-06-29

    CD44 is a broadly distributed cell surface protein thought to mediate cell attachment to extracelular matrix components or specific cell surface ligands. We have created soluble CD44-immunoglobulin fusion proteins and characterized their reactivity with tissue sections and lymph node high endothelial cells in primary culture. The CD44 target on high endothelial cells is sensitive to enzymes that degrade hyaluronate, and binding of soluble CD44 is blocked by low concentrations of hyaluronate or high concentrations of chondroitin 4- and 6-sulfates. A mouse anti-hamster hyaluonate receptor antibody reacts with COS cells expressing hamster CD44 cDNA. In sections of all tissues examined, including lymph nodes and Peyer's patches, predigestion with hyaluronidase eliminated CD44 binding.

  16. Heptapeptide ligands against receptor-binding sites of influenza hemagglutinin toward anti-influenza therapy.

    Science.gov (United States)

    Matsubara, Teruhiko; Onishi, Ai; Yamaguchi, Daisuke; Sato, Toshinori

    2016-03-01

    The initial attachment of influenza virus to cells is the binding of hemagglutinin (HA) to the sialyloligosaccharide receptor; therefore, the small molecules that inhibit the sugar-protein interaction are promising as HA inhibitors to prevent the infection. We herein demonstrate that sialic acid-mimic heptapeptides are identified through a selection from a primary library against influenza virus HA. In order to obtain lead peptides, an affinity selection from a phage-displayed random heptapeptide library was performed with the HAs of the H1 and H3 strains, and two kinds of the HA-binding peptides were identified. The binding of the peptides to HAs was inhibited in the presence of sialic acid, and plaque assays indicated that the corresponding N-stearoyl peptide strongly inhibited infections by the A/Aichi/2/68 (H3N2) strain of the virus. Alanine scanning of the peptides indicated that arginine and proline were responsible for binding. The affinities of several mutant peptides with single-amino-acid substitutions against H3 HA were determined, and corresponding docking studies were performed. A Spearman analysis revealed a correlation between the affinity of the peptides and the docking study. These results provide a practicable method to design of peptide-based HA inhibitors that are promising as anti-influenza drugs.

  17. Non-peptide ligand binding to the formyl peptide receptor FPR2--A comparison to peptide ligand binding modes.

    Science.gov (United States)

    Stepniewski, Tomasz M; Filipek, Slawomir

    2015-07-15

    Ligands of the FPR2 receptor initiate many signaling pathways including activation of phospholipase C, protein kinase C, the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/protein kinase B pathway. The possible actions include also calcium flux, superoxide generation, as well as migration and proliferation of monocytes. FPR2 activation may induce a pro- and anti-inflammatory effect depending on the ligand type. It is also found that this receptor is involved in tumor growth. Most of currently known FPR2 ligands are agonists since they were designed based on N-formyl peptides, which are natural agonists of formyl receptors. Since the non-peptide drugs are indispensable for effective treatment strategies, we performed a docking study of such ligands employing a generated dual template homology model of the FPR2 receptor. The study revealed different binding modes of particular classes of these drugs. Based on the obtained docking poses we proposed a detailed location of three hydrophobic pockets in orthosteric binding site of FPR2. Our model emphasizes the importance of aromatic stacking, especially with regard to residues His102(3.29) and Phe257(6.51), for binding of FPR2 ligands. We also identified other residues important for non-peptide ligand binding in the binding site of FPR2. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

    Science.gov (United States)

    Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

    1992-01-01

    The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

  19. Binding characteristics of sigma2 receptor ligands Características estruturais de ligantes do receptor sigma2

    Directory of Open Access Journals (Sweden)

    Richard A. Glennon

    2005-03-01

    Full Text Available Sigma (sigma receptors, once considered a type of opioid receptor, are now recognized as representing a unique receptive entity and at least two different types of sigma receptors have been identified: sigma1 and sigma2 receptors. Evidence suggests that these receptors might be targeted and exploited for the development of agents potentially useful for the treatment of several central disorders. This review primarily describes some of our efforts to understand those structural features that contribute to sigma2 receptor binding, and some recent work by other investigators is also included. Despite an inability to formulate a unified pharmacophore model for sigma2 binding due to the diversity of structure-types that bind at the receptor, and to the conformational flexibility of these ligands, significant progress has been made toward the development of some very high-affinity agents.Receptores sigma (sigma, considerados como um tipo de receptor opióide, sigma ão hoje considerados como uma entidade receptora singular. Pelo menos dois subtipos desses receptores foram identificados: sigma1e sigma2. Há evidências de que esses receptores devam ser explorados como alvo para o desenvolvimento de agentes potencialmente úteis para o tratamento de várias disfunções centrais. Esta revisão descreve, principalmente, alguns dos nossos esforços para compreender as características estruturais que contribuem para a ligação no receptor sigma2 , e incluem-se alguns trabalhos recentes desenvolvidos por outros pesquisadores. Apesar da incapacidade de formular um modelo de farmacóforo único para ligação no receptor s 2, em razão da diversidade de estruturas que a ele se ligam e da flexibilidade conformacional desses ligantes, houve progresso significativo no desenvolvimento de agentes de alta afinidade.

  20. The human olfactory receptor 17-40: requisites for fitting into the binding pocket.

    Science.gov (United States)

    Anselmi, Cecilia; Buonocore, Anna; Centini, Marisanna; Facino, Roberto Maffei; Hatt, Hanns

    2011-06-01

    To gain structural insight on the interactions between odorants and the human olfactory receptor, we did homology modelling of the receptor structure, followed by molecular docking simulation with ligands. Molecular dynamics simulation on the structures resulting from docking served to estimate the binding free energy of the various odorant families. A correlation with the odorous properties of the ligands is proposed. We also investigated which residues were involved in the binding of a set of properly synthesised ligands and which were required for fitting inside the binding pocket. Olfactive stimulation of the olfactory receptor with odorous molecules was also investigated, using calcium imaging or electrophysiological recordings.

  1. Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG.

    Science.gov (United States)

    Kyakumoto, S; Kurokawa, R; Ota, M

    1990-07-12

    Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor

  2. Effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza A (H3N2) vaccine viruses.

    Science.gov (United States)

    Parker, Lauren; Wharton, Stephen A; Martin, Stephen R; Cross, Karen; Lin, Yipu; Liu, Yan; Feizi, Ten; Daniels, Rodney S; McCauley, John W

    2016-06-01

    Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.

  3. Analysis of the Binding Sites of Porcine Sialoadhesin Receptor with PRRSV

    Directory of Open Access Journals (Sweden)

    Yibo Jiang

    2013-12-01

    Full Text Available Porcine reproductive and respiratory syndrome virus (PRRSV can infect pigs and cause enormous economic losses to the pig industry worldwide. Porcine sialoadhesin (pSN and CD163 have been identified as key viral receptors on porcine alveolar macrophages (PAM, a main target cell infected by PRRSV. In this study, the protein structures of amino acids 1–119 from the pSN and cSN (cattle sialoadhesin N-termini (excluding the 19-amino acid signal peptide were modeled via homology modeling based on mSN (mouse sialoadhesin template structures using bioinformatics tools. Subsequently, pSN and cSN homology structures were superposed onto the mSN protein structure to predict the binding sites of pSN. As a validation experiment, the SN N-terminus (including the wild-type and site-directed-mutant-types of pSN and cSN was cloned and expressed as a SN-GFP chimera protein. The binding activity between SN and PRRSV was confirmed by WB (Western blotting, FAR-WB (far Western blotting, ELISA (enzyme-linked immunosorbent assay and immunofluorescence assay. We found that the S107 amino acid residue in the pSN N-terminal played a crucial role in forming a special cavity, as well as a hydrogen bond for enhancing PRRSV binding during PRRSV infection. S107 may be glycosylated during PRRSV infection and may also be involved in forming the cavity for binding PRRSV along with other sites, including W2, Y44, S45, R97, R105, W106 and V109. Additionally, S107 might also be important for pSN binding with PRRSV. However, the function of these binding sites must be confirmed by further studies.

  4. Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

    Science.gov (United States)

    Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

    2012-12-04

    Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

  5. High throughput techniques for discovering new glycine receptor modulators and their binding sites

    Directory of Open Access Journals (Sweden)

    Daniel F Gilbert

    2009-10-01

    Full Text Available The inhibitory glycine receptor (GlyR is a member of the Cys-loop receptor family that mediates inhibitory neurotransmission in the central nervous system. These receptors are emerging as potential drug targets for inflammatory pain, immunomodulation, spasticity and epilepsy. Antagonists that specifically inhibit particular GlyR isoforms are also required as pharmacological probes for elucidating the roles of particular GlyR isoforms in health and disease. Although a substantial number of both positive and negative GlyR modulators have been identified, very few of these are specific for the GlyR over other receptor types. Thus, the potential of known compounds as either therapeutic leads or pharmacological probes is limited. It is therefore surprising that there have been few published studies describing attempts to discover novel GlyR isoform-specific compounds. The first aim of this review is to consider various methods for efficiently screening compounds against these receptors. We conclude that an anion sensitive yellow fluorescent protein is optimal for primary screening and that automated electrophysiology of cells stably expressing GlyRs is useful for confirming hits and quantitating the actions of identified compounds. The second aim of this review is to demonstrate how these techniques are used in our laboratory for the purpose of both discovering novel GlyR-active compounds and characterizing their binding sites. We also describe a reliable, cost effective method for transfecting HEK293 cells in single wells of a 384 well plate using nanogram quantities of cDNA.

  6. The evolution of natural killer cell receptors.

    Science.gov (United States)

    Carrillo-Bustamante, Paola; Keşmir, Can; de Boer, Rob J

    2016-01-01

    Natural killer (NK) cells are immune cells that play a crucial role against viral infections and tumors. To be tolerant against healthy tissue and simultaneously attack infected cells, the activity of NK cells is tightly regulated by a sophisticated array of germline-encoded activating and inhibiting receptors. The best characterized mechanism of NK cell activation is "missing self" detection, i.e., the recognition of virally infected or transformed cells that reduce their MHC expression to evade cytotoxic T cells. To monitor the expression of MHC-I on target cells, NK cells have monomorphic inhibitory receptors which interact with conserved MHC molecules. However, there are other NK cell receptors (NKRs) encoded by gene families showing a remarkable genetic diversity. Thus, NKR haplotypes contain several genes encoding for receptors with activating and inhibiting signaling, and that vary in gene content and allelic polymorphism. But if missing-self detection can be achieved by a monomorphic NKR system why have these polygenic and polymorphic receptors evolved? Here, we review the expansion of NKR receptor families in different mammal species, and we discuss several hypotheses that possibly underlie the diversification of the NK cell receptor complex, including the evolution of viral decoys, peptide sensitivity, and selective MHC-downregulation.

  7. Receptors for T cell-replacing factor/interleukin 5. Specificity, quantitation, and its implication

    OpenAIRE

    1988-01-01

    T cell-replacing factor (TRF)/IL-5 is a glycosylated polypeptide that acts as a key factor for B cell growth and differentiation. Since IL-5 action is probably mediated by specific cell surface receptor(s), we have characterized the binding of IL-5 to cells using biosynthetically [35S]methionine-labeled IL-5 and 125I-IL-5 that had been prepared using Bolton-Hunter reagent. The radiolabeled IL-5 binds specifically to BCL1- B20 (in vitro line) (a murine chronic B cell leukemic cell line previou...

  8. Analyzing machupo virus-receptor binding by molecular dynamics simulations

    Directory of Open Access Journals (Sweden)

    Austin G. Meyer

    2014-02-01

    Full Text Available In many biological applications, we would like to be able to computationally predict mutational effects on affinity in protein–protein interactions. However, many commonly used methods to predict these effects perform poorly in important test cases. In particular, the effects of multiple mutations, non alanine substitutions, and flexible loops are difficult to predict with available tools and protocols. We present here an existing method applied in a novel way to a new test case; we interrogate affinity differences resulting from mutations in a host–virus protein–protein interface. We use steered molecular dynamics (SMD to computationally pull the machupo virus (MACV spike glycoprotein (GP1 away from the human transferrin receptor (hTfR1. We then approximate affinity using the maximum applied force of separation and the area under the force-versus-distance curve. We find, even without the rigor and planning required for free energy calculations, that these quantities can provide novel biophysical insight into the GP1/hTfR1 interaction. First, with no prior knowledge of the system we can differentiate among wild type and mutant complexes. Moreover, we show that this simple SMD scheme correlates well with relative free energy differences computed via free energy perturbation. Second, although the static co-crystal structure shows two large hydrogen-bonding networks in the GP1/hTfR1 interface, our simulations indicate that one of them may not be important for tight binding. Third, one viral site known to be critical for infection may mark an important evolutionary suppressor site for infection-resistant hTfR1 mutants. Finally, our approach provides a framework to compare the effects of multiple mutations, individually and jointly, on protein–protein interactions.

  9. A cation-pi interaction in the binding site of the glycine receptor is mediated by a phenylalanine residue

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Millen, Kat S; Hanek, Ariele P;

    2008-01-01

    Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-pi interaction with the agonist, whereas in GABA(A) receptors, a Tyr performs this role. The glycine receptor binding site, however, contains pr...

  10. Molecular dissection of the intrinsic factor-vitamin B12 receptor, cubilin, discloses regions important for membrane association and ligand binding.

    Science.gov (United States)

    Kristiansen, M; Kozyraki, R; Jacobsen, C; Nexø, E; Verroust, P J; Moestrup, S K

    1999-07-16

    Cubilin, the receptor for intrinsic factor-vitamin B12, is a novel type of high molecular weight receptor consisting of a 27 CUB (complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domain cluster preceded by 8 epidermal growth factor repeats and a short N-terminal sequence. In addition to binding the vitamin B12-carrier complex, cubilin also binds receptor-associated protein. To delineate the structures for membrane association and ligand binding we established a panel of stable transfected Chinese hamster ovary cells expressing overlapping segments of rat cubilin. Analysis of conditioned media and cell extracts of transfected cells revealed that the N-terminal cubilin region conveys membrane association. Helical plotting of this region demonstrated a conserved amphipathic helix pattern (Lys74-Glu109) as a candidate site for hydrophobic interactions. Ligand affinity chromatography and surface plasmon resonance analysis of the secreted cubilin fragments showed ligand binding in the CUB domain region. Further dissection of binding-active fragments localized the binding site for intrinsic factor-vitamin B12 to CUB domains 5-8 and a receptor-associated protein-binding site to CUB domains 13-14. In conclusion, the N-terminal cubilin region seems crucial for membrane association, whereas the CUB domain cluster harbors distinct sites for ligand binding.

  11. Binding of quinolizidine alkaloids to nicotinic and muscarinic acetylcholine receptors.

    Science.gov (United States)

    Schmeller, T; Sauerwein, M; Sporer, F; Wink, M; Müller, W E

    1994-09-01

    Fourteen quinolizidine alkaloids, isolated from Lupinus albus, L. mutabilis, and Anagyris foetida, were analyzed for their affinity for nicotinic and/or muscarinic acetylcholine receptors. Of the compounds tested, the alpha-pyridones, N-methylcytisine and cytisine, showed the highest affinities at the nicotinic receptor, while several quinolizidine alkaloid types were especially active at the muscarinic receptor.

  12. Familial Risk for Major Depression is Associated with Lower Striatal 5-HT4 Receptor Binding

    DEFF Research Database (Denmark)

    Madsen, Karine; Torstensen, Eva; Holst, Klaus Kähler

    2015-01-01

    BACKGROUND: The 5-HT4 receptor provides a novel potential target for antidepressant treatment. No studies exist to elucidate the 5-HT4 receptor's in vivo distribution in the depressed state or in populations that may display trait markers for major depression disorder (MDD). The aim of this study......-degree relatives with a history of MDD binding correlated negatively with 5-HT4 receptor binding in both the striatum (p = 0.001) and limbic regions (p = 0.012). CONCLUSIONS: Our data suggest that the 5-HT4 receptor is involved in the neurobiological mechanism underlying familial risk for depression...

  13. Mu receptor binding of some commonly used opioids and their metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhaorong; Irvine, R.J. (Univ. of Adelaide (Australia)); Somogyi, A.A.; Bochner, F. (Univ. of Adelaide (Australia) Royal Adelaide Hospital (Australia))

    1991-01-01

    The binding affinity to the {mu} receptor of some opioids chemically related to morphine and some of their metabolites was examined in rat brain homogenates with {sup 3}H-DAMGO. The chemical group at position 6 of the molecule had little effect on binding. Decreasing the length of the alkyl group at position 3 decreased the K{sub i} values (morphine < codeine < ethylmorphine < pholcodine). Analgesics with high clinical potency containing a methoxyl group at position 3 had relatively weak receptor binding, while their O-demethylated metabolites had much stronger binding. Many opioids may exert their pharmacological actions predominantly through metabolites.

  14. In vitro binding of a radio-labeled positive allosteric modulator for metabotropic glutamate receptor subtype 5.

    Science.gov (United States)

    Zysk, John R; Spear, Nathan; Fieles, William; Stein, Mark M; Sygowski, Linda S; King, Megan M; Hoesch, Valerie; Hastings, Richard; Brockel, Becky; Do, Mylinh; Ström, Peter; Gadient, Reto; Chhajlani, Vijay; Elmore, Charles S; Maier, Donna L

    2013-03-01

    The positive allosteric modulator (PAM) binding site for metabotropic glutamate receptor subtype 5 (mGlu(5)) lacks a readily available radio-labeled tracer fordetailed structure-activity studies. This communication describes a selective mGlu(5) compound, 7-methyl-2-(4-(pyridin-2-yloxy)benzyl)-5-(pyridin-3-yl)isoindolin-1-one (PBPyl) that binds with high affinity to human mGlu(5) and exhibits functional PAM activity. Analysis of PBPyl by FLIPR revealed an EC(50) of 87 nM with an 89% effect in transfected HEK293 cells and an EC(50) of 81 nM with a 42% effect in rat primary neurons. PBPyl exhibited 5-fold higher functional selectivity for mGlu(5) in a full mGlu receptor panel. Unlabeled PBPyl was tested for specific binding using a liquid chromatography mass spectrometry (LC/MS/MS)-based filtration binding assay and exhibited 40% specific binding in recombinant membranes, a value higher than any candidate compound tested. In competition binding studies with [(3)H]MPEP, the mGlu(5) receptor negative allosteric modulator (NAM), PBPyl exhibited a k(i) value of 34 nM. PBPyl also displaced [(3)H]ABP688, a mGluR(5) receptor NAM, in tissue sections from mouse and rat brain using autoradiography. Areas of specific binding included the frontal cortex, striatum and nucleus accumbens. PBPyl was radiolabeled to a specific activity of 15 Ci/mmol and tested for specific binding in a filter plate format. In recombinant mGlu(5b) membranes, [(3)H] PBPyl exhibited saturable binding with a K(d) value of 18.6 nM. In competition binding experiments, [(3)H] PBPyl was displaced by high affinity mGlu(5) positive and negative modulators. Further tests showed that PBPyl displays less than optimal characteristics as an in vivo tool, including a high volume of distribution and ClogP, making it more suitable as an in vitro compound. However, as a first report of direct binding of an mGlu(5) receptor PAM, this study offers value toward the development of novel PET imaging agents for this important

  15. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    .05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...

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

    Directory of Open Access Journals (Sweden)

    Marc P Maillard

    2001-03-01

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

  17. Identification of the Calmodulin-Binding Domains of Fas Death Receptor.

    Directory of Open Access Journals (Sweden)

    Bliss J Chang

    Full Text Available The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD and that of the Fas-associated protein (FADD interact to form the core of the death-inducing signaling complex (DISC, a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD. However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR, biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas-mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209-239 (Fas-Pep1 and 251-288 (Fas-Pep2 constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD-CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling

  18. A single-molecule approach to explore binding, uptake and transport of cancer cell targeting nanotubes

    Science.gov (United States)

    Lamprecht, C.; Plochberger, B.; Ruprecht, V.; Wieser, S.; Rankl, C.; Heister, E.; Unterauer, B.; Brameshuber, M.; Danzberger, J.; Lukanov, P.; Flahaut, E.; Schütz, G.; Hinterdorfer, P.; Ebner, A.

    2014-03-01

    In the past decade carbon nanotubes (CNTs) have been widely studied as a potential drug-delivery system, especially with functionality for cellular targeting. Yet, little is known about the actual process of docking to cell receptors and transport dynamics after internalization. Here we performed single-particle studies of folic acid (FA) mediated CNT binding to human carcinoma cells and their transport inside the cytosol. In particular, we employed molecular recognition force spectroscopy, an atomic force microscopy based method, to visualize and quantify docking of FA functionalized CNTs to FA binding receptors in terms of binding probability and binding force. We then traced individual fluorescently labeled, FA functionalized CNTs after specific uptake, and created a dynamic ‘roadmap’ that clearly showed trajectories of directed diffusion and areas of nanotube confinement in the cytosol. Our results demonstrate the potential of a single-molecule approach for investigation of drug-delivery vehicles and their targeting capacity.

  19. Effect of ethanol administration and withdrawal on GABA receptor binding in rat cerebral cortex

    Energy Technology Data Exchange (ETDEWEB)

    Volicer, L.; Biagioni, T.M.

    1982-01-01

    Sodium independent GABA receptor binding was measured in synaptosomes prepared from cerebral cortex of rats made ethanol dependent by three daily ethanol administrations. In rats sacrificed 1 hour after the last ethanol dose there was a lower number of low affinity binding sites and lower affinity of the high affinity binding than in controls. The decreased affinity was present only in rats who showed symptoms of ethanol withdrawal during the course of ethanol administration. In rats sacrificed during ethanol withdrawal the affinity of the high affinity binding was lower than in controls and other binding characteristics were unchanged. This decreased binding was normalized by repeated Triton X-100 incubations indicating involvement of an endogenous inhibitor in this ethanol effect. Acute ethanol administration did not change GABA receptor binding.

  20. CLiBE: a database of computed ligand binding energy for ligand-receptor complexes.

    Science.gov (United States)

    Chen, X; Ji, Z L; Zhi, D G; Chen, Y Z

    2002-11-01

    Consideration of binding competitiveness of a drug candidate against natural ligands and other drugs that bind to the same receptor site may facilitate the rational development of a candidate into a potent drug. A strategy that can be applied to computer-aided drug design is to evaluate ligand-receptor interaction energy or other scoring functions of a designed drug with that of the relevant ligands known to bind to the same binding site. As a tool to facilitate such a strategy, a database of ligand-receptor interaction energy is developed from known ligand-receptor 3D structural entries in the Protein Databank (PDB). The Energy is computed based on a molecular mechanics force field that has been used in the prediction of therapeutic and toxicity targets of drugs. This database also contains information about ligand function and other properties and it can be accessed at http://xin.cz3.nus.edu.sg/group/CLiBE.asp. The computed energy components may facilitate the probing of the mode of action and other profiles of binding. A number of computed energies of some PDB ligand-receptor complexes in this database are studied and compared to experimental binding affinity. A certain degree of correlation between the computed energy and experimental binding affinity is found, which suggests that the computed energy may be useful in facilitating a qualitative analysis of drug binding competitiveness.

  1. Characterization of ( sup 3 H)alprazolam binding to central benzodiazepine receptors

    Energy Technology Data Exchange (ETDEWEB)

    McCabe, R.T.; Mahan, D.R.; Smith, R.B.; Wamsley, J.K. (Neuropsychiatric Research Institute, Fargo, ND (USA))

    1990-10-01

    The binding of the triazolobenzodiazepine ({sup 3}H)alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Studies using nonequilibrium and equilibrium binding conditions for ({sup 3}H)alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of ({sup 3}H)alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. The Kd was 4.6 nM and the Bmax was 2.6 pmol/mg protein. GABA enhanced ({sup 3}H)alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bind to other receptor sites had a very weak or negligible effect on ({sup 3}H)alprazolam binding. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. The biochemical binding profile does not appear to account for the unique therapeutic properties which distinguish this compound from the other benzodiazepines in its class.

  2. Influence of the hinge region and its adjacent domains on binding and signaling patterns of the thyrotropin and follitropin receptor.

    Directory of Open Access Journals (Sweden)

    Jörg Schaarschmidt

    Full Text Available Glycoprotein hormone receptors (GPHR have a large extracellular domain (ECD divided into the leucine rich repeat (LRR domain for binding of the glycoprotein hormones and the hinge region (HinR, which connects the LRR domain with the transmembrane domain (TMD. Understanding of the activation mechanism of GPHRs is hindered by the unknown interaction of the ECD with the TMD and the structural changes upon ligand binding responsible for receptor activation. Recently, our group showed that the HinR of the thyrotropin receptor (TSHR can be replaced by those of the follitropin (FSHR and lutropin receptor (LHCGR without effects on surface expression and hTSH signaling. However, differences in binding characteristics for bovine TSH at the various HinRs were obvious. To gain further insights into the interplay between LRR domain, HinR and TMD we generated chimeras between the TSHR and FSHR. Our results obtained by the determination of cell surface expression, ligand binding and G protein activation confirm the similar characteristics of GPHR HinRs but they also demonstrate an involvement of the HinR in ligand selectivity indicated by the observed promiscuity of some chimeras. While the TSHR HinR contributes to specific binding of TSH and its variants, no such contribution is observed for FSH and its analog TR4401 at the HinR of the FSHR. Furthermore, the charge distribution at the poorly characterized LRR domain/HinR transition affected ligand binding and signaling even though this area is not in direct contact with the ligand. In addition our results also demonstrate the importance of the TMD/HinR interface. Especially the combination of the TSHR HinR with the FSHR-TMD resulted in a loss of cell surface expression of the respective chimeras. In conclusion, the HinRs of GPHRs do not only share similar characteristics but also behave as ligand specific structural and functional entities.

  3. Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation.

    Science.gov (United States)

    Chen, Shaoyong; Gulla, Sarah; Cai, Changmeng; Balk, Steven P

    2012-03-01

    Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

  4. Tumor cell marker PVRL4 (nectin 4 is an epithelial cell receptor for measles virus.

    Directory of Open Access Journals (Sweden)

    Ryan S Noyce

    2011-08-01

    Full Text Available Vaccine and laboratory adapted strains of measles virus can use CD46 as a receptor to infect many human cell lines. However, wild type isolates of measles virus cannot use CD46, and they infect activated lymphocytes, dendritic cells, and macrophages via the receptor CD150/SLAM. Wild type virus can also infect epithelial cells of the respiratory tract through an unidentified receptor. We demonstrate that wild type measles virus infects primary airway epithelial cells grown in fetal calf serum and many adenocarcinoma cell lines of the lung, breast, and colon. Transfection of non-infectable adenocarcinoma cell lines with an expression vector encoding CD150/SLAM rendered them susceptible to measles virus, indicating that they were virus replication competent, but lacked a receptor for virus attachment and entry. Microarray analysis of susceptible versus non-susceptible cell lines was performed, and comparison of membrane protein gene transcripts produced a list of 11 candidate receptors. Of these, only the human tumor cell marker PVRL4 (Nectin 4 rendered cells amenable to measles virus infections. Flow cytometry confirmed that PVRL4 is highly expressed on the surfaces of susceptible lung, breast, and colon adenocarcinoma cell lines. Measles virus preferentially infected adenocarcinoma cell lines from the apical surface, although basolateral infection was observed with reduced kinetics. Confocal immune fluorescence microscopy and surface biotinylation experiments revealed that PVRL4 was expressed on both the apical and basolateral surfaces of these cell lines. Antibodies and siRNA directed against PVRL4 were able to block measles virus infections in MCF7 and NCI-H358 cancer cells. A virus binding assay indicated that PVRL4 was a bona fide receptor that supported virus attachment to the host cell. Several strains of measles virus were also shown to use PVRL4 as a receptor. Measles virus infection reduced PVRL4 surface expression in MCF7 cells, a

  5. The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Nemčovičová, Ivana [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States); Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava (Slovakia); Zajonc, Dirk M., E-mail: dzajonc@liai.org [La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 (United States)

    2014-03-01

    The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155 as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor

  6. 几种阿片类药物在表达人μ阿片受体Sf9昆虫细胞中的受体亲和力及依赖性%Binding affinity to and dependence on some opioids in Sf9 insect cells expressing humanμ-opioid receptor

    Institute of Scientific and Technical Information of China (English)

    刘忠华; 和友; 金文桥; 陈新建; 张鸿萍; 申庆祥; 池志强

    2003-01-01

    目的:用表达人μ受体的Sf9昆虫细胞(Sf9-μ细胞)研究二氢埃托啡、芬太尼、海洛因和哌替啶对μ受体亲和力与依赖性.方法:竞争结合实验检测受体亲和力;放免法测定cAMP;热板法测痛;纳洛酮催促跳跃评价身体依赖性.结果:Sf9-μ细胞经上述药物孵育后,纳洛酮引起cAMP超射.以结合实验中Ki值与cAMP超射实验中的EC50值之比作为药物在Sf9-μ细胞的依赖性指数.以镇痛ED50与戒断反应中累积剂量的ED50之比作为小鼠身体依赖性指数.上述药物的两种依赖性指数之间以及受体亲和力与镇痛活性之间有较好的相关性.结论:Sf9-μ细胞可用来评价阿片类药物的受体亲和力及依赖潜能.%AIM: To investigate the receptor binding affinity and naloxone-precipitated cAMP overshoot of dihydroetorphine,fentanyl, heroin, and pethidine in Sf9 insect cells expressing human μ-opioid receptor (Sf9-μ cells). METHODS:Competitive binding assay of [3H]ohmefentanyl was used to reveal the affinity for μ-opioid receptor in Sf9-μ cells.[3H]cAMP RIA was used to determine cAMP level. Antinociceptive activity was evaluated using 55 ℃ mouse hot plate test. Naloxone-precipitated withdrawal jumping was used to reflect physical dependence in mice. RESULTS:All drugs displayed antinociceptive activity and produced physical dependence in mice. The Ki values of dihydroetorphine, fentanyl, heroin, and pethidine in competitive binding assay were (0.85±0.20) nmol, (59.1± 11.7)nmol, (0.36±0.13) μmol, and (12.2±3.8) μmol respectively. The binding affinities of these drugs for μ-opioid receptor in Sf9-μ cells were paralleled to their antinociceptive activities in mice. After chronic pretreatment with these drugs, naloxone induced cAMP withdrawal overshoot in Sf9-μ cells. The dependence index in Sf9-μ cells was calculated as Ki value in competitive binding assay over ECs0 value in naloxone-precipitated cAMP assay. The physical dependence index in mice

  7. Mast Cell and Immune Inhibitory Receptors

    Institute of Scientific and Technical Information of China (English)

    LixinLi; ZhengbinYao

    2004-01-01

    Modulation by balancing activating and inhibitory receptors constitutes an important mechanism for regulating immune responses. Cells that are activated following ligation of receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs) can be negatively regulated by other receptors bearing immunoreceptor tyrosine-based inhibition motifs (ITIMs). Human mast cells (MCs) are the major effector cells of type I hypersensitivity and important participants in a number of disease processes. Antigen-mediated aggregation of IgE bound to its high-affinity receptor on MCs initiates a complex series of biochemical events leading to MC activation. With great detailed description and analysis of several inhibitory receptors on human MCs, a central paradigm of negative regulation of human MC activation by these receptors has emerged. Cellular & Molecular Immunology. 2004;1(6):408-415.

  8. Neutralization of biological activity and inhibition of receptor binding by antibodies against human thrombopoietin.

    Science.gov (United States)

    Tahara, T; Kuwaki, T; Matsumoto, A; Morita, H; Watarai, H; Inagaki, Y; Ohashi, H; Ogami, K; Miyazaki, H; Kato, T

    1998-01-01

    Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D8 to Q28, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S1 to R117 and A60 to K122 of hTPO, indicating that the epitope(s) of TN1 is localized in residues A60 to R117 of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S47 to D62, L108 to A126, N172 to A190, S262 to T284, and P306 to G332 of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D8 to Q28 and A60 to R117 of hTPO, may contain the domains associated with its receptor, C-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.

  9. A propofol binding site on mammalian GABAA receptors identified by photolabeling

    Science.gov (United States)

    Yip, Grace M S; Chen, Zi-Wei; Edge, Christopher J; Smith, Edward H; Dickinson, Robert; Hohenester, Erhard; Townsend, R Reid; Fuchs, Karoline; Sieghart, Werner; Evers, Alex S; Franks, Nicholas P

    2014-01-01

    Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA receptors, but where it binds to this receptor is not known and has been a matter of some controversy. We have synthesized a novel propofol analogue photolabeling reagent that has a biological activity very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin which have been identified using X-ray crystallography. Using a combination of the protiated label and a deuterated version, and mammalian receptors labeled in intact membranes, we have identified a novel binding site for propofol in GABAA receptors consisting of both β3 homopentamers and α1β3 heteropentamers. The binding site is located within the β subunit, at the interface between the transmembrane domains and the extracellular domain, and lies close to known determinants of anesthetic sensitivity in transmembrane segments TM1 and TM2. PMID:24056400

  10. Binding kinetics of membrane-anchored receptors and ligands: Molecular dynamics simulations and theory.

    Science.gov (United States)

    Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard; Weikl, Thomas R

    2015-12-28

    The adhesion of biological membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. Central questions are how the binding kinetics of these proteins is affected by the membranes and by the membrane anchoring of the proteins. In this article, we (i) present detailed data for the binding of membrane-anchored proteins from coarse-grained molecular dynamics simulations and (ii) provide a theory that describes how the binding kinetics depends on the average separation and thermal roughness of the adhering membranes and on the anchoring, lengths, and length variations of the proteins. An important element of our theory is the tilt of bound receptor-ligand complexes and transition-state complexes relative to the membrane normals. This tilt results from an interplay of the anchoring energy and rotational entropy of the complexes and facilitates the formation of receptor-ligand bonds at membrane separations smaller than the preferred separation for binding. In our simulations, we have considered both lipid-anchored and transmembrane receptor and ligand proteins. We find that the binding equilibrium constant and binding on-rate constant of lipid-anchored proteins are considerably smaller than the binding constant and on-rate constant of rigid transmembrane proteins with identical binding domains.

  11. ( sup 3 H)cytisine binding to nicotinic cholinergic receptors in brain

    Energy Technology Data Exchange (ETDEWEB)

    Pabreza, L.A.; Dhawan, S.; Kellar, K.J. (Georgetown Univ. School of Medicine, Washington, DC (USA))

    1991-01-01

    Cytisine, a ganglionic agonist, competes with high affinity for brain nicotinic cholinergic receptors labeled by any of several nicotinic {sup 3}H-agonist ligands. Here we have examined the binding of ({sup 3}H)cytisine in rat brain homogenates. ({sup 3}H)Cytisine binds with high affinity (Kd less than 1 nM), and specific binding represented 60-90% of total binding at all concentrations examined up to 15 nM. The nicotinic cholinergic agonists nicotine, acetylcholine, and carbachol compete with high affinity for ({sup 3}H)cytisine binding sites, whereas among nicotinic receptor antagonists only dihydro-beta-erythroidine competes with high affinity (in the nanomolar range). Comparison of binding in several brain regions showed that ({sup 3}H)cytisine binding is higher in the thalamus, striatum, and cortex than in the hippocampus, cerebellum, or hypothalamus. The pharmacology and brain regional distribution of ({sup 3}H)cytisine binding sites are those predicted for neuronal nicotinic receptor agonist recognition sites. The high affinity and low nonspecific binding of ({sup 3}H)cytisine shoul