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Sample records for aberrant receptor binding

  1. Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3

    TAKAOKA, MUNENORI; Harada, Hideki; Andl, Claudia D; Oyama, Kenji; Naomoto, Yoshio; Dempsey, Kelly L.; Klein-Szanto, Andres J.; El-Deiry, Wafik S; GRIMBERG, ADDA; Nakagawa, Hiroshi

    2004-01-01

    Epidermal growth factor receptor (EGFR) is frequently overexpressed in esophageal carcinoma and its precursor lesions. To gain insights into how EGFR overexpression affects cellular functions in primary human esophageal cells, we performed gene expression profiling and identified insulin-like growth factor-binding protein (IGFBP)-3 as the most up-regulated gene. IGFBP-3 regulates cell proliferation through both insulin-like growth factor-dependent and independent mechanisms. We found that IGF...

  2. Clinicopathological significance of aberrant Notch receptors in intrahepatic cholangiocarcinoma

    Wu, Wen-Rui; Shi, Xiang-De; Zhang, Rui; Zhu, Man-Sheng; Xu, Lei-Bo; Yu, Xian-Huan; Zeng, Hong; Wang, Jie; Liu, Chao

    2014-01-01

    Notch signaling has been reported to be activated to promote biliary epithelial cell differentiation and tubulogenesis during bile duct development. In this study, clinicopathological significance of aberrant expression of Notch receptors in intrahepatic cholangiocarcinoma (ICC) was investigated. Thus, forty-one ICC specimens were examined by immunohistochemistry using anti-Notch1-4 antibodies, respectively. Expression of Notch receptors was scored by percentage of positive tumor cells and intensity of immunostaining. Clinicopathological parameters and survival data were compared with the expression of Notch receptors, respectively. Expression of Notch receptors was identified in cancer cells, as well as in non-neoplastic cells. Compared with adjacent non-tumor liver tissues, Notch1 and 4 were up regulated, and Notch2 and 3 were relatively weaker. Positive immunostaining of Notch1 in ICC cells was detected in 34 cases (82.9%), Notch2 in 23 (56.1%), Notch3 in 16 (39.0%) and Notch4 in 14 (34.1%). Notch1 was overexpressed in cases with tumor size > 5 cm (P = 0.036). Expression of Notch2 was correlated inversely with histological grade (P = 0.016). Overexpression of Notch4 was more common in cases with serum CA125 > 35 U/ml than cases with CA125 ≤ 35 U/ml (P = 0.048). Expression of Notch3 was not correlated with any other clinicopathological parameters. Moreover, Notch4 was related to poor survival (P < 0.001). To conclude, this study reveals that aberrant expression of Notch receptors 1 and 4 might play important roles during ICC progression. PMID:25031748

  3. Radioligand Binding at Muscarinic Receptors

    El-Fakahany, E. E.; Jakubík, Jan

    New York: Springer, 2016 - (Mysliveček, J.; Jakubík, J.), s. 37-68. (Neuromethods. 107). ISBN 978-1-4939-2857-6 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptors * radioligand binding Subject RIV: ED - Physiology

  4. Glucocorticoid receptor transformation and DNA binding

    The overall goal is to probe the mechanism whereby glucocorticoid receptors are transformed from a non-DNA-binding form to their active DNA-binding form. The author has examined the effect of an endogenous inhibitor purified from rat liver cytosol on receptor binding to DNA. The inhibitor binds to transformed receptors in whole cytosol and prevent their binding to DNA. He also examined the role of sulfhydryl groups in determining the DNA binding activity of the transformed receptor and in determining the transformation process. Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 00C with the sulfhydryl modifying reagent methyl methanethiosulfonate inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol. In addition, he has examined the relationship between receptor phosphorylation and DNA binding. Untransformed receptor complexes purified from cytosol prepared from mouse L cells grown in medium containing [32P]orthophosphate contain two components, a 100 k-Da and a 90-kDa subunit, both of which are phosphoproteins. On transformation, the receptor dissociates from the 90-kDa protein. Transformation of the complex under cell free conditions does not result in a dephosphorylation of the 100-kDa steroid-binding protein. Transformed receptor that has been bound to DNA and purified by monoclonal antibody is still in a phosphorylated form. These results suggest that dephosphorylation is not required for receptor binding to DNA

  5. Identical splicing of aberrant epidermal growth factor receptor transcripts from amplified rearranged genes in human glioblastomas.

    Sugawa, N; Ekstrand, A J; James, C D; Collins, V P

    1990-01-01

    The epidermal growth factor receptor gene has been found to be amplified and rearranged in human glioblastomas in vivo. Here we present the sequence across a splice junction of aberrant epidermal growth factor receptor transcripts derived from corresponding and uniquely rearranged genes that are coamplified and coexpressed with non-rearranged epidermal growth factor receptor genes in six primary human glioblastomas. Each of these six tumors contains aberrant transcripts derived from identical...

  6. Impact of receptor clustering on ligand binding

    Caré Bertrand R

    2011-03-01

    Full Text Available Abstract Background Cellular response to changes in the concentration of different chemical species in the extracellular medium is induced by ligand binding to dedicated transmembrane receptors. Receptor density, distribution, and clustering may be key spatial features that influence effective and proper physical and biochemical cellular responses to many regulatory signals. Classical equations describing this kind of binding kinetics assume the distributions of interacting species to be homogeneous, neglecting by doing so the impact of clustering. As there is experimental evidence that receptors tend to group in clusters inside membrane domains, we investigated the effects of receptor clustering on cellular receptor ligand binding. Results We implemented a model of receptor binding using a Monte-Carlo algorithm to simulate ligand diffusion and binding. In some simple cases, analytic solutions for binding equilibrium of ligand on clusters of receptors are provided, and supported by simulation results. Our simulations show that the so-called "apparent" affinity of the ligand for the receptor decreases with clustering although the microscopic affinity remains constant. Conclusions Changing membrane receptors clustering could be a simple mechanism that allows cells to change and adapt its affinity/sensitivity toward a given stimulus.

  7. Binding characteristics of swine erythrocyte insulin receptors

    Crossbred gilts had 8.8 +/- 1.1% maximum binding of [125I]insulin to insulin receptors on erythrocytes. The number of insulin-binding sites per cell was 137 +/- 19, with a binding affinity ranging from 7.4 X 10(7)M-1 to 11.2 X 10(7)M-1 and mean of 8.8 X 10(7)M-1. Pregnant sows had a significant increase in maximum binding due to an increase in number of receptor sites per cell. Lactating sows fed a high-fiber diet and a low-fiber diet did not develop a significant difference in maximum binding of insulin. Sows fed the low-fiber diet had a significantly higher number of binding sites and a significantly lower binding affinity than did sows fed a high-fiber diet. Receptor-binding affinity was lower in the low-fiber diet group than in cycling gilts, whereas data from sows fed the high-fiber diet did not differ from data for cycling gilts. Data from this study indicated that insulin receptors of swine erythrocytes have binding characteristics similar to those in other species. Pregnancy and diet will alter insulin receptor binding in swine

  8. Receptor binding studies of the living heart

    Receptors form a class of intrinsic membrane proteins (or glycoproteins) defined by the high affinity and specificity with which they bind ligands. Many receptors are associated directly or indirectly with membrane ion channels that open or close after a conformational change of the receptor induced by the binding of the neurotransmitter. Changes in number and/or affinity of cardiac neurotransmitter receptors have been associated with myocardial ischemia and infarction, congestive heart failure, and cardiomyopathy as well as diabetes or thyroid-induced heart muscle disease. These alterations of cardiac receptors have been demonstrated in vitro on membrane homogenates from samples collected mainly during surgery or postmortem. The disadvantage of these in vitro binding techniques is that receptors lose their natural environment and their relationships with the other components of the tissue

  9. Binding of Glutamate to the Umami Receptor

    Lopez Cacales, J.; Oliviera Costa, S.; de Groot, B.; Walters, D

    2010-01-01

    Abstract The umami taste receptor is a heterodimer composed of two members of the T1R taste receptor family: T1R1 and T1R3. It detects glutamate in humans, and is a more general amino acid detector in other species. We have constructed homology models of the ligand binding domains of the human umami receptor (based on crystallographic structures of the metabotropic glutamate receptor of the central nervous system). We have carried out molecular dynamics simulations of the ligand bi...

  10. Receptor binding studies of soft anticholinergic agents

    Huang, Fenglei; Buchwald, Peter; Browne, Clinton E.; Farag, Hassan H.; Wu, Wnei-Mei; Ji, Fubao; Hochhaus, Guenther; Bodor, Nicholas

    2001-01-01

    Receptor binding studies were performed on 24 soft anticholinergic agents and 5 conventional anticholinergic agents using 4 cloned human muscarinic receptor subtypes. The measured pKi values of the soft anticholinergic agents ranged from 6.5 to 9.5, with the majority being in the range of 7.5 to 8.5. Strong correlation was observed between the pKis determined here and the pA2 values measured earlier in guinea pig ileum contraction assays. The corresponding correlation coefficients (r2) were 0...

  11. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    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.

  12. Bladder endothelin-1 receptor binding of bosentan and ambrisentan.

    Osano, Ayaka; Yokoyama, Yoshinari; Hayashi, Hideki; Itoh, Kunihiko; Okura, Takashi; Deguchi, Yoshiharu; Ito, Yoshihiko; Yamada, Shizuo

    2014-01-01

    The present study aimed to characterize bladder endothelin-1 (ET-1) receptor binding of clinically used ET-1 receptor antagonists by using [(125)I]ET-1. The inhibition of specific [(125)I]ET-1 binding was measured in the presence of ET-1 and its receptor antagonists. Specific binding of [(125)I]ET-1 in rat bladder was saturable and of high affinity, which characterized selective labeling of bladder ET-1 receptors. ET-1, bosentan, ambrisentan, and CI-1020 inhibited specific [(125)I]ET-1 binding in a concentration-dependent manner at nanomolar ranges of IC50. Nonlinear least squares regression analysis revealed the presence of high- and low-affinity ET-1 receptor sites for ambrisentan and CI-1020. Bosentan and ambrisentan significantly increased the dissociation constant for bladder [(125)I]ET-1 binding without affecting maximal number of binding sites (Bmax). Thus, bosentan and ambrisentan seem to bind to bladder ET-1 receptor in a competitive and reversible manner. Oral administration of bosentan caused a dose-dependent decrease in Bmax for bladder [(125)I]ET-1 binding, suggesting significant binding of bladder ET-1 receptors in vivo. A significant amount of pharmacologically relevant ET-1 receptors may exist in the bladder. These receptors may be implicated in the pathogenesis of lower urinary tract symptoms and may also be promising targets for the development of therapeutic agents. PMID:24389822

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

    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

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

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

    2016-01-01

    -like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), respectively. To define CGRP receptor binding sites, in vitro autoradiography was performed with [(3)H]MK-3207 (a CGRP receptor antagonist). CLR and RAMP1 mRNA and protein expression were detected in the pineal gland, medial mammillary...

  15. Characterization of pulmonary sigma receptors by radioligand binding.

    Lever, John R; Litton, Tyler P; Fergason-Cantrell, Emily A

    2015-09-01

    This study establishes the expression of appreciable populations of sites on mouse lung membranes that exhibit radioligand binding properties and pharmacology consistent with assignment as sigma1 and sigma2 receptors. Specific binding of the sigma1 receptor radioligand [(3)H](+)-pentazocine reached steady state within 6h at 37°C. Saturation studies revealed high affinity binding to a single class of sites (Kd 1.36±0.04nM; Bmax 967±11fmol/mg protein). Inhibition studies showed appropriate sigma1 receptor pharmacology, including higher affinity for (+)-N-allylnormetazocine with respect to the (-)-enantiomer, and positive allosteric modulation of dextromethorphan binding by phenytoin. Using [(3)H]1,3-di(2-tolyl)guanidine in the presence of (+)-pentazocine to assess sigma2 receptor binding, steady state was achieved within 2min at 25°C. Cold saturation studies revealed one high affinity, low capacity binding site (Kd 31.8±8.3nM; Bmax 921±228fmol/mg protein) that displayed sigma2 receptor pharmacology. A very low affinity, high capacity interaction also was observed that represents saturable, but not sigma receptor specific, binding. A panel of ligands showed rank order inhibition of radioligand binding appropriate for the sigma2 receptor, with ifenprodil displaying the highest apparent affinity. In vivo, dextromethorphan inhibited the specific binding of a radioiodinated sigma1 receptor ligand in lung with an ED50 of 1.2μmol/kg, a value near the recommended dosage for the drug as a cough suppressant. Overall, the present work provides a foundation for studies of drug interactions with pulmonary sigma1 and sigma2 receptors in vitro and in vivo. PMID:26004528

  16. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

    Meyyammai Swaminathan

    2014-06-01

    Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

  17. Risperidone treatment increases CB1 receptor binding in rat brain

    Secher, Anna; Husum, Henriette; Holst, Birgitte;

    2010-01-01

    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....... 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...

  18. Characterisation of the melanocortin 4 receptor by radioligand binding

    The DNA encoding the human melanocortin 4 receptor was expressed in COS (CV-1 origin, Sv 40) cells and its radioligand binding properties was tested by using the [124I[(Nle4, D-Phe7) αmelanocyte stimulating hormone (MSH). The radioligand was found to bind to a single saturable site with a Kd of 3l84±0.57 nmol/l in the MC4 receptor expressing cells. The order of potency of a number of substance competing for the [1225I[[Nle4, D-Phe7[ αMSH binding was the following; [Nle4, D-Phe7[ α-MSH>[Nlee[-α-MSH>β-MSH>desacetyl-α-MSH >α-MSH>ACTH (1-39)>ACTH (4-10)>γ2-MSH. This order of potency is unique for the melanocortin 4 receptor when compared to our previously published data for the other melanocortin receptor subtypes. Most notably the melanocortin 4 receptor shows highest affinity for β-MSH, among the endogenous MSH-peptides. Furthermore the melanocortin 4 receptor shows very low affinity for the γ-MSH peptides. This distinguishes the melanocortin 4 receptor from the melanocortin 3 receptor, which is the other major central nervous system melanocortin-receptor, as melanocortin 3 receptor shows high affinity for γ-MSH. Our finding might indicate a specific role for β-MSH for the melanocortin 4 receptor. (au) 31 refs

  19. Deletion of glutamate delta-1 receptor in mouse leads to aberrant emotional and social behaviors.

    Roopali Yadav

    Full Text Available The delta family of ionotropic glutamate receptors consists of glutamate δ1 (GluD1 and glutamate δ2 (GluD2 receptors. While the role of GluD2 in the regulation of cerebellar physiology is well understood, the function of GluD1 in the central nervous system remains elusive. We demonstrate for the first time that deletion of GluD1 leads to abnormal emotional and social behaviors. We found that GluD1 knockout mice (GluD1 KO were hyperactive, manifested lower anxiety-like behavior, depression-like behavior in a forced swim test and robust aggression in the resident-intruder test. Chronic lithium rescued the depression-like behavior in GluD1 KO. GluD1 KO mice also manifested deficits in social interaction. In the sociability test, GluD1 KO mice spent more time interacting with an inanimate object compared to a conspecific mouse. D-Cycloserine (DCS administration was able to rescue social interaction deficits observed in GluD1 KO mice. At a molecular level synaptoneurosome preparations revealed lower GluA1 and GluA2 subunit expression in the prefrontal cortex and higher GluA1, GluK2 and PSD95 expression in the amygdala of GluD1 KO. Moreover, DCS normalized the lower GluA1 expression in prefrontal cortex of GluD1 KO. We propose that deletion of GluD1 leads to aberrant circuitry in prefrontal cortex and amygdala owing to its potential role in presynaptic differentiation and synapse formation. Furthermore, these findings are in agreement with the human genetic studies suggesting a strong association of GRID1 gene with several neuropsychiatric disorders including schizophrenia, bipolar disorder, autism spectrum disorders and major depressive disorder.

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

    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 receptor revealed that the anion is bound between the peptide rings of the biscyclopeptide. Intimate intramolecular contacts between the nonpolar surfaces of the proline rings of the individual recep...

  1. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

    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; Harold A. Flores Quintana; 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 s...

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

    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.

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

    RobertRoot-Bernstein

    2014-07-01

    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.

  4. In Silico Investigation of the Neurotensin Receptor 1 Binding Site

    Lückmann, Michael; Holst, Birgitte; Schwartz, Thue W.;

    2016-01-01

    The neurotensin receptor 1 (NTSR1) belongs to the family of 7TM, G protein-coupled receptors, and is activated by the 13-amino-acid peptide neurotensin (NTS) that has been shown to play important roles in neurol. disorders and the promotion of cancer cells. Recently, a high-resoln. x-ray crystal...... structure of NTSR1 in complex with NTS8-13 has been detd., providing novel insights into peptide ligand recognition by 7TM receptors. SR48692, a potent and selective small mol. antagonist has previously been used extensively as a tool compd. to study NTSR1 receptor signaling properties. To investigate...... the binding mode of SR48692 and other small mol. compds. to NTSR1, we applied an Automated Ligand-guided Backbone Ensemble Receptor Optimization protocol (ALiBERO), taking receptor flexibility and ligand knowledge into account. Structurally overlapping binding poses for SR48692 and NTS8-13 were obsd., despite...

  5. Fatty acid binding receptors in intestinal physiology and pathophysiology

    Kaemmerer, Elke; Plum, Patrick; Klaus, Christina; Weiskirchen, Ralf; Liedtke, Christian; Adolf, Maximilian; Schippers, Angela; Wagner, Norbert; Reinartz, Andrea; Gassler, Nikolaus

    2010-01-01

    Free fatty acids are essential dietary components and recognized as important molecules in the maintenance of cellular homeostasis. In the last decade, the molecular pathways for free fatty acid sensing in the gastrointestinal tract have been further elucidated by molecular identification and functional characterization of fatty acid binding receptors. These sensing molecules belong to the family of G protein-coupled receptors. In the intestine, four important receptors have been described so...

  6. σ1 Receptor Modulation of G-Protein-Coupled Receptor Signaling: Potentiation of Opioid Transduction Independent from Receptor Binding

    Kim, Felix J.; Kovalyshyn, Ivanka; Burgman, Maxim; Neilan, Claire; Chien, Chih-Cheng

    2010-01-01

    σ Ligands modulate opioid actions in vivo, with agonists diminishing morphine analgesia and antagonists enhancing the response. Using human BE(2)-C neuroblastoma cells that natively express opioid receptors and human embryonic kidney (HEK) cells transfected with a cloned μ opioid receptor, we now demonstrate a similar modulation of opioid function, as assessed by guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding, by σ1 receptors. σ Ligands do not compete opioid receptor binding. Administered alone, neither σ agonists nor antagonists significantly stimulated [35S]GTPγS binding. Yet σ receptor selective antagonists, but not agonists, shifted the EC50 of opioid-induced stimulation of [35S]GTPγS binding by 3- to 10-fold to the left. This enhanced potency was seen without a change in the efficacy of the opioid, as assessed by the maximal stimulation of [35S]GTPγS binding. σ1 Receptors physically associate with μ opioid receptors, as shown by coimmunoprecipitation studies in transfected HEK cells, implying a direct interaction between the proteins. Thus, σ receptors modulate opioid transduction without influencing opioid receptor binding. RNA interference knockdown of σ1 in BE(2)-C cells also potentiated μ opioid-induced stimulation of [35S]GTPγS binding. These modulatory actions are not limited to μ and δ opioid receptors. In mouse brain membrane preparations, σ1-selective antagonists also potentiated both opioid receptor and muscarinic acetylcholine receptor-mediated stimulation of [35S]GTPγS binding, suggesting a broader role for σ receptors in modulating G-protein-coupled receptor signaling. PMID:20089882

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

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

    2010-04-01

    sigma Ligands modulate opioid actions in vivo, with agonists diminishing morphine analgesia and antagonists enhancing the response. Using human BE(2)-C neuroblastoma cells that natively express opioid receptors and human embryonic kidney (HEK) cells transfected with a cloned mu opioid receptor, we now demonstrate a similar modulation of opioid function, as assessed by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding, by sigma(1) receptors. sigma Ligands do not compete opioid receptor binding. Administered alone, neither sigma agonists nor antagonists significantly stimulated [(35)S]GTP gamma S binding. Yet sigma receptor selective antagonists, but not agonists, shifted the EC(50) of opioid-induced stimulation of [(35)S]GTP gamma S binding by 3- to 10-fold to the left. This enhanced potency was seen without a change in the efficacy of the opioid, as assessed by the maximal stimulation of [(35)S]GTP gamma S binding. sigma(1) Receptors physically associate with mu opioid receptors, as shown by coimmunoprecipitation studies in transfected HEK cells, implying a direct interaction between the proteins. Thus, sigma receptors modulate opioid transduction without influencing opioid receptor binding. RNA interference knockdown of sigma(1) in BE(2)-C cells also potentiated mu opioid-induced stimulation of [(35)S]GTP gamma S binding. These modulatory actions are not limited to mu and delta opioid receptors. In mouse brain membrane preparations, sigma(1)-selective antagonists also potentiated both opioid receptor and muscarinic acetylcholine receptor-mediated stimulation of [(35)S]GTP gamma S binding, suggesting a broader role for sigma receptors in modulating G-protein-coupled receptor signaling. PMID:20089882

  8. Effect of desipramine on dopamine receptor binding in vivo

    Effect of desipramine on the in vivo binding of 3H-SCH23390 and 3H-N-methylspiperone (3H-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 3H-SCH23390 or 45 min after injection of 3H-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

  9. Modeling of ligand binding to dopamine D2 receptor

    Ostopovici-Halip Liliana

    2014-01-01

    Full Text Available The dopaminic receptors have been for long time the major targets for developing new small molecules with high affinity and selectivity to treat psychiatric disorders, neurodegeneration, drug abuse, and other therapeutic areas. In the absence of a 3D structure for the human D2 dopamine (HDD2 receptor, the efforts for discovery and design of new potential drugs rely on comparative models generation, docking and pharmacophore development studies. To get a better understanding of the HDD2 receptor binding site and the ligand-receptor interactions a homology model of HDD2 receptor based on the X-ray structure of β2-adrenergic receptor has been built and used to dock a set of partial agonists of HDD2 receptor. The main characteristics of the binding mode for the HDD2 partial agonists set are given by the ligand particular folding and a complex network of contacts represented by stacking interactions, salt bridge and hydrogen bond formation. The characterization of the partial agonist binding mode at HDD2 receptor provide the needed information to generate pharmacophore models which represent essential information in the future virtual screening studies in order to identify new potential HDD2 partial agonists.

  10. Characterisation of the melanocortin 4 receptor by radioligand binding

    Schioeth, H.B.; Wikberg, J.E.S. [Uppsala Univ., Dept. of Pharmaceutical Pharmacology, Uppsala (Sweden); Muceniece, R. [Inst. of Organic Synthesis, Lab. of Pharmacology, Riga (Latvia)

    1996-09-01

    The DNA encoding the human melanocortin 4 receptor was expressed in COS (CV-1 origin, Sv 40) cells and its radioligand binding properties was tested by using the [{sup 124}I](Nle{sup 4}, D-Phe{sup 7}) {alpha}melanocyte stimulating hormone (MSH). The radioligand was found to bind to a single saturable site with a K{sub d} of 3l84{+-}0.57 nmol/l in the MC4 receptor expressing cells. The order of potency of a number of substance competing for the [{sup 12}25I][Nle{sup 4}, D-Phe{sup 7}] {alpha}MSH binding was the following; [Nle{sup 4}, D-Phe{sup 7}] {alpha}-MSH>[Nle{sup e}]-{alpha}-MSH>{beta}-MSH>desacetyl-{alpha}-MSH >{alpha}-MSH>ACTH (1-39)>ACTH (4-10)>{gamma}2-MSH. This order of potency is unique for the melanocortin 4 receptor when compared to our previously published data for the other melanocortin receptor subtypes. Most notably the melanocortin 4 receptor shows highest affinity for {beta}-MSH, among the endogenous MSH-peptides. Furthermore the melanocortin 4 receptor shows very low affinity for the {gamma}-MSH peptides. This distinguishes the melanocortin 4 receptor from the melanocortin 3 receptor, which is the other major central nervous system melanocortin-receptor, as melanocortin 3 receptor shows high affinity for {gamma}-MSH. Our finding might indicate a specific role for {beta}-MSH for the melanocortin 4 receptor. (au) 31 refs.

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

    RobertRoot-Bernstein

    2014-01-01

    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 recepto...

  12. Coregulator Control of Androgen Receptor Action by a Novel Nuclear Receptor-Binding Motif

    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

    2014-01-01

    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 rec...

  13. Differential effect of glucocorticoid receptor antagonists on glucocorticoid receptor nuclear translocation and DNA binding

    Spiga, Francesca; Knight, David M; Droste, Susanne K; Conway-Campbell, Becky; Kershaw, Yvonne; MacSweeney, Cliona P; Thomson, Fiona J; Craighead, Mark; Peeters, Bernard WMM; Lightman, Stafford L

    2016-01-01

    The effects of RU486 and S-P, a more selective glucocorticoid receptor antagonist from Schering-Plough, were investigated on glucocorticoid receptor nuclear translocation and DNA binding. In the in vitro study, AtT20 cells were treated with vehicle or with RU486, S-P or corticosterone (3–300 nM) or co-treated with vehicle or glucocorticoid receptor antagonists (3–300 nM) and 30 nM corticosterone. Both glucocorticoid receptor antagonists induced glucocorticoid receptor nuclear translocation but only RU486 induced DNA binding. RU486 potentiated the effect of corticosterone on glucocorticoid receptor nuclear translocation and DNA binding, S-P inhibited corticosterone-induced glucocorticoid receptor nuclear translocation, but not glucocorticoid receptor-DNA binding. In the in vivo study, adrenalectomized rats were treated with vehicle, RU486 (20 mg/kg) and S-P (50 mg/kg) alone or in combination with corticosterone (3 mg/kg). RU486 induced glucocorticoid receptor nuclear translocation in the pituitary, hippocampus and prefrontal cortex and glucocorticoid receptor-DNA binding in the hippocampus, whereas no effect of S-P on glucocorticoid receptor nuclear translocation or DNA binding was observed in any of the areas analysed. These findings reveal differential effects of RU486 and S-P on areas involved in regulation of hypothalamic–pituitary–adrenal axis activity in vivo and they are important in light of the potential use of this class of compounds in the treatment of disorders associated with hyperactivity of the hypothalamic–pituitary–adrenal axis. PMID:20093322

  14. Binding Mode Prediction of Evodiamine within Vanilloid Receptor TRPV1

    Huaping Liang; Liangren Zhang; Wuzhuang Gong; Yanhui Zhang; Zhanli Wang; Lidan Sun; Hui Yu; Hongwei Jin

    2012-01-01

    Accurate assessment of the potential binding mode of drugs is crucial to computer-aided drug design paradigms. It has been reported that evodiamine acts as an agonist of the vanilloid receptor Transient receptor potential vanilloid-1 (TRPV1). However, the precise interaction between evodiamine and TRPV1 was still not fully understood. In this perspective, the homology models of TRPV1 were generated using the crystal structure of the voltage-dependent shaker family K

  15. Structural Allostery and Binding of the Transferring Receptor Complex

    Xu,G.; Liu, R.; Zak, O.; Aisen, P.; Chance, M.

    2005-01-01

    The structural allostery and binding interface for the human serum transferrin (Tf){center_dot}transferrin receptor (TfR) complex were identified using radiolytic footprinting and mass spectrometry. We have determined previously that the transferrin C-lobe binds to the receptor helical domain. In this study we examined the binding interactions of full-length transferrin with receptor and compared these data with a model of the complex derived from cryoelectron microscopy (cryo-EM) reconstructions. The footprinting results provide the following novel conclusions. First, we report characteristic oxidations of acidic residues in the C-lobe of native Tf and basic residues in the helical domain of TfR that were suppressed as a function of complex formation; this confirms ionic interactions between these protein segments as predicted by cryo-EM data and demonstrates a novel method for detecting ion pair interactions in the formation of macromolecular complexes. Second, the specific side-chain interactions between the C-lobe and N-lobe of transferrin and the corresponding interactions sites on the transferrin receptor predicted from cryo-EM were confirmed in solution. Last, the footprinting data revealed allosteric movements of the iron binding C- and N-lobes of Tf that sequester iron as a function of complex formation; these structural changes promote tighter binding of the metal ion and facilitate efficient ion transport during endocytosis.

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

    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 125I-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. 125I-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

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

    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...

  18. Development of a homogeneous binding assay for histamine receptors.

    Crane, Kathy; Shih, Daw-Tsun

    2004-12-01

    Histamine is critically involved in a wide range of physiological and pathological processes through its actions at different receptors. Thus, histamine receptors have been actively pursued as therapeutic targets in the pharmaceutical industry for the treatment of a variety of diseases. There are currently four histamine receptors that have been cloned, all of which are G protein-coupled receptors. Studies from both academia and pharmaceutical companies have identified compounds that modulate the function of specific histamine receptors. These efforts led to the successful introduction of histamine H(1) and H(2) receptor antagonists for the treatment of allergy and excess gastric acid secretion, respectively. Histamine H(3) receptor ligands are currently under investigation for the treatment of obesity and neurological disorders. The recently identified histamine H(4) receptor is preferentially expressed in the immune tissues, suggesting a potential role in normal immune functions and possibly in the pathogenesis of inflammatory diseases. Even with the long history of histamine research and the important applications of histamine receptor ligands, assays to measure the affinity of compounds binding to histamine receptors are still routinely analyzed using a filtration assay, a very low-throughput assay involving washing and filtration steps. This article describes a simple, robust, and homogeneous binding assay based on the scintillation proximity assay (SPA) technology that provides results equivalent to those obtained using the more complex filtration assay. The SPA format is easily adapted to high-throughput screening because it is amenable to automation. In summary, this technique allows high-throughput screening of compounds against multiple histamine receptors and, thus, facilitates drug discovery efforts. PMID:15519569

  19. The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands

    Repič, Matej; Zakšek, Maja; Kotnik, Kristina; Fijan, Estera; Mavri, Janez

    2016-01-01

    In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N–H and O–H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure. PMID:27159606

  20. The receptor binding domain of botulinum neurotoxin serotype C binds phosphoinositides.

    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 LD(50) of ∼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. Using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro domain. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides in both assays. Interactions with phosphoinositides may facilitate tighter binding between neuronal membranes and BoNT/C. PMID:22120109

  1. Whole-genome cartography of estrogen receptor alpha binding sites.

    Chin-Yo Lin

    2007-06-01

    Full Text Available Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript, suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs, 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha

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

    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.

  3. Blocking and binding folate receptor alpha autoantibodies identify novel autism spectrum disorder subgroups

    Richard Eugene Frye

    2016-03-01

    Full Text Available Folate receptor α (FRα autoantibodies (FRAAs are prevalent in autism spectrum disorder (ASD. They disrupt the transportation of folate across the blood-brain barrier by binding to the FRα. Children with ASD with FRAAs have been reported to respond well to treatment with a form of folate known as folinic acid, suggesting that they may be an important ASD subgroup to identify and treat. There has been no investigation of whether they manifest unique behavioral and physiological characteristics. Thus, in this study we measured both blocking and binding FRAAs, physiological measurements including indices of redox and methylation metabolism and inflammation as well as serum folate and B12 concentrations and measurements of development and behavior in 94 children with ASD. Children positive for the binding FRAA were found to have higher serum B12 levels as compared to those negative for binding FRAAs while children positive for the blocking FRAA were found to have relatively better redox metabolism and inflammation markers as compared to those negative for blocking FRAAs. In addition, ASD children positive for the blocking FRAA demonstrated better communication on the Vineland Adaptive Behavior Scale, stereotyped behavior on the Aberrant Behavioral Checklist and Mannerisms on the Social Responsiveness Scale. This study suggests that FRAAs are associated with specific physiological and behavioral characteristics in children with ASD and provides support for the notion that these biomarkers may be useful for subgrouping children with ASD, especially with respect to targeted treatments.

  4. Blocking and Binding Folate Receptor Alpha Autoantibodies Identify Novel Autism Spectrum Disorder Subgroups.

    Frye, Richard E; Delhey, Leanna; Slattery, John; Tippett, Marie; Wynne, Rebecca; Rose, Shannon; Kahler, Stephen G; Bennuri, Sirish C; Melnyk, Stepan; Sequeira, Jeffrey M; Quadros, Edward

    2016-01-01

    Folate receptor α (FRα) autoantibodies (FRAAs) are prevalent in autism spectrum disorder (ASD). They disrupt the transportation of folate across the blood-brain barrier by binding to the FRα. Children with ASD and FRAAs have been reported to respond well to treatment with a form of folate known as folinic acid, suggesting that they may be an important ASD subgroup to identify and treat. There has been no investigation of whether they manifest unique behavioral and physiological characteristics. Thus, in this study we measured both blocking and binding FRAAs, physiological measurements including indices of redox and methylation metabolism and inflammation as well as serum folate and B12 concentrations and measurements of development and behavior in 94 children with ASD. Children positive for the binding FRAA were found to have higher serum B12 levels as compared to those negative for binding FRAAs while children positive for the blocking FRAA were found to have relatively better redox metabolism and inflammation markers as compared to those negative for blocking FRAAs. In addition, ASD children positive for the blocking FRAA demonstrated better communication on the Vineland Adaptive Behavior Scale, stereotyped behavior on the Aberrant Behavioral Checklist and mannerisms on the Social Responsiveness Scale. This study suggests that FRAAs are associated with specific physiological and behavioral characteristics in children with ASD and provides support for the notion that these biomarkers may be useful for subgrouping children with ASD, especially with respect to targeted treatments. PMID:27013943

  5. D-2 receptor binding in dopa-responsive dystonia

    Kunig, G; Leenders, KL; Antonini, A; Vontobel, P; Weindl, A; Meinck, HM

    1998-01-01

    We have studied dopamine D-2 receptor binding by [C-11]raclopride positron emission tomography in 14 patients with dopa-responsive dystonia (DRD). Data were compared with 16 levodopa-treated patients with Parkinson's disease (PD) and 26 healthy controls. The results revealed an elevated [C-11]raclop

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

    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.

  7. In vitro radioprotective effects of histamine H2 receptor antagonists against gamma-rays induced chromosomal aberrations in human lymphocytes

    Background: Radioprotective capability of histamine H2 receptor antagonists have been shown in several in vivo studies mainly using animal models. However, to verify the effectiveness of these agents in clinical applications, studies should be performed on human lymphocytes using metaphase analysis. Materials and Methods: In vitro metaphase analysis technique was used to test the effects of cimetidine, ranitidine and famotidine on radiation induced clastogenic effects. Lymphocytes in whole peripheral blood were exposed to 3 Gy gamma-rays at a dose rate of 73.7 c Gy/min in the presence or absence of various doses of the drugs used in this study. The frequency of chromosomal aberrations were determined after standard metaphase preparations and staining slides in 5% Giemsa. Results: Results show that radiation produced a high number of chromosomal aberrations in lymphocytes compared to controls (p2-receptor antagonists used in this study effectively reduced the clastogenic effects of radiation with a dose reduction factor of 1.5-2 in human lymphocytes in vitro. The way in which these drugs reduce the clastogenic effects of radiation might be via radical scavenging mechanism

  8. Evidence for an intrinsic binding force between dodecaborate dianions and receptors with hydrophobic binding pockets.

    Warneke, Jonas; Jenne, Carsten; Bernarding, Johannes; Azov, Vladimir A; Plaumann, Markus

    2016-05-01

    A gas phase binding study revealed strong intrinsic intermolecular interactions between dianionic halogenated closo-dodecaborates [B12X12](2-) and several neutral organic receptors. Oxidation of a tetrathiafulvalene host allowed switching between two host-guest binding modes in a supramolecular complex. Complexes of β-cyclodextrin with [B12F12](2-) show remarkable stability in the gas phase and were successfully tested as carriers for the delivery of boron clusters into cancer cells. PMID:27087168

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

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

    2015-01-01

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

  10. ERPs Reveal the Time-Course of Aberrant Visual-Phonological Binding in Developmental Dyslexia

    Jones, Manon W.; Kuipers, Jan-Rouke; Thierry, Guillaume

    2016-01-01

    New evidence is accumulating for a deficit in binding visual-orthographic information with the corresponding phonological code in developmental dyslexia. Here, we identify the mechanisms underpinning this deficit using event-related brain potentials (ERPs) in dyslexic and control adult readers performing a letter-matching task. In each trial, a printed letter was presented synchronously with an auditory letter name. Incongruent (mismatched), frequent trials were interleaved with congruent (matched) infrequent target pairs, which participants were asked to report by pressing a button. In critical trials, incongruent letter pairs were mismatched but confusable in terms of their visual or phonological features. Typical readers showed early detection of deviant trials, indicated by larger modulation in the range of the phonological mismatch negativity (PMN) compared with standard trials. This was followed by stronger modulation of the P3b wave for visually confusable deviants and an increased lateralized readiness potential (LRP) for phonological deviants, compared with standards. In contrast, dyslexic readers showed reduced sensitivity to deviancy in the PMN range. Responses to deviants in the P3b range indicated normal letter recognition processes, but the LRP calculation revealed a specific impairment for visual-orthographic information during response selection in dyslexia. In a follow-up experiment using an analogous non-lexical task in the same participants, we found no reading-group differences, indicating a degree of specificity to over-learnt visual-phonological binding. Our findings indicate early insensitivity to visual-phonological binding in developmental dyslexia, coupled with difficulty selecting the correct orthographic code. PMID:26973493

  11. ERPs reveal the time-course of aberrant visual-phonological binding in developmental dyslexia

    Manon Wyn Jones

    2016-03-01

    Full Text Available New evidence is accumulating for a deficit in binding visual-orthographic information with the corresponding phonological code in developmental dyslexia. Here, we identify the mechanisms underpinning this deficit using event-related brain potentials (ERPs in dyslexic and control adult readers performing a letter-matching task. In each trial, a printed letter was presented synchronously with an auditory letter name. Incongruent (mismatched, frequent trials were interleaved with congruent (matched infrequent target pairs, which participants were asked to report by pressing a button. In critical trials, incongruent letter pairs were mismatched but confusable in terms of their visual or phonological features. Typical readers showed early detection of deviant trials, indicated by larger modulation in the range of the phonological mismatch negativity (PMN compared with standard trials. This was followed by stronger modulation of the P3b wave for visually confusable deviants and an increased lateralized readiness potential (LRP for phonological deviants, compared with standards. In contrast, dyslexic readers showed reduced sensitivity to deviancy in the PMN range. Responses to deviants in the P3b range indicated normal letter recognition processes, but the LRP calculation revealed a specific impairment for visual-orthographic information during response selection in dyslexia. In a follow-up experiment using an analogous non-lexical task in the same participants, we found no reading-group differences, indicating a degree of specificity to over-learnt visual-phonological binding. Our findings indicate early insensitivity to visual-phonological binding in developmental dyslexia, coupled with difficulty selecting the correct orthographic code.

  12. ERPs Reveal the Time-Course of Aberrant Visual-Phonological Binding in Developmental Dyslexia.

    Jones, Manon W; Kuipers, Jan-Rouke; Thierry, Guillaume

    2016-01-01

    New evidence is accumulating for a deficit in binding visual-orthographic information with the corresponding phonological code in developmental dyslexia. Here, we identify the mechanisms underpinning this deficit using event-related brain potentials (ERPs) in dyslexic and control adult readers performing a letter-matching task. In each trial, a printed letter was presented synchronously with an auditory letter name. Incongruent (mismatched), frequent trials were interleaved with congruent (matched) infrequent target pairs, which participants were asked to report by pressing a button. In critical trials, incongruent letter pairs were mismatched but confusable in terms of their visual or phonological features. Typical readers showed early detection of deviant trials, indicated by larger modulation in the range of the phonological mismatch negativity (PMN) compared with standard trials. This was followed by stronger modulation of the P3b wave for visually confusable deviants and an increased lateralized readiness potential (LRP) for phonological deviants, compared with standards. In contrast, dyslexic readers showed reduced sensitivity to deviancy in the PMN range. Responses to deviants in the P3b range indicated normal letter recognition processes, but the LRP calculation revealed a specific impairment for visual-orthographic information during response selection in dyslexia. In a follow-up experiment using an analogous non-lexical task in the same participants, we found no reading-group differences, indicating a degree of specificity to over-learnt visual-phonological binding. Our findings indicate early insensitivity to visual-phonological binding in developmental dyslexia, coupled with difficulty selecting the correct orthographic code. PMID:26973493

  13. Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules

    Xu, Lifeng; Blackburn, Elizabeth H.

    2004-01-01

    We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No...

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

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    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...... 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...

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

    Høyer-Hansen, G; Behrendt, N; Ploug, M; Danø, K; Preissner, K T

    1997-01-01

    -blotting experiments we found that vitronectin binds uPAR but not uPAR(2+3). In real-time biomolecular interaction analysis using recombinant, soluble uPAR (suPAR) both plasma and multimeric forms of vitronectin bound to intact, antibody-immobilized suPAR. Monoclonal antibodies against domain 1 of uPAR blocked su......PAR binding to vitronectin and vitronectin did not interact with suPAR(2+3). Both suPAR(2+3) and the isolated domain 1 failed to compete with the intact suPAR in binding to vitronectin. We therefore conclude that the intact receptor is required for efficient vitronectin binding....

  16. Aberrant splicing of androgenic receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity

    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symtpoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. The authors report a G → T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46, XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein ∼5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo

  17. Receptor binding of biosynthetic human insulin on isolated pig hepatocytes

    Gammeltoft, S.

    Biosynthetic human insulin (BHI) and pancreatic human insulin were compared with respect to receptor binding in a heterologous assay system: displacement of pork A14-/sup 125/I-monoiodoinsulin from receptors on pig hepatocytes. The concentrations of human insulin giving half-maximal displacement were identical for both preparations, i.e., 0.5 nM. Their relative potency was 1.01 +/- 0.14 (SD, N . 5), suggesting that biosynthetic and pancreatic human insulin exert the same biologic activity.

  18. Receptor binding of biosynthetic human insulin on isolated pig hepatocytes

    Biosynthetic human insulin (BHI) and pancreatic human insulin were compared with respect to receptor binding in a heterologous assay system: displacement of pork A14-125I-monoiodoinsulin from receptors on pig hepatocytes. The concentrations of human insulin giving half-maximal displacement were identical for both preparations, i.e., 0.5 nM. Their relative potency was 1.01 +/- 0.14 (SD, N . 5), suggesting that biosynthetic and pancreatic human insulin exert the same biologic activity

  19. Binding of polychlorinated biphenyls to the aryl hydrocarbon receptor.

    Kafafi, S A; Afeefy, H Y; A. H. Ali; Said, H K; Kafafi, A G

    1993-01-01

    A new thermodynamic model for calculating the dissociation constants of complexes formed between the aryl hydrocarbon receptor (AhR) and polychlorinated biphenyls (PCBs) is reported. The free energies of binding of PCBs to AhR are controlled by their lipophilicities, electron affinities, and entropies. The corresponding physicochemical properties of polychlorinated dibenzo-p-dioxins and dibenzofurans also control their interactions with AhR. We present evidence supporting the hypothesis that ...

  20. Binding Mode of Insulin Receptor and Agonist Peptide

    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

  1. Aberrant splicing of androgen receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity.

    Ris-Stalpers, C.; Kuiper, G G; Faber, P.W.; SCHWEIKERT, H. U.; van Rooij, H C; Zegers, N.D.; Hodgins, M B; Degenhart, H J; Trapman, J; Brinkmann, A.O.

    1990-01-01

    Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symptoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. We report a G----T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46,XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely a...

  2. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  3. Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells

  4. Cholinergic receptor binding in the frontal cortex of suicide victims

    Because there is a high incidence of individuals diagnosed as having an affective disorder who subsequently commit suicide, the author thought it would be of interest to determine QNB binding in the brains of a large sample of suicide victims, and to compare the findings with a well-matched control group. Brain samples were obtained at autopsy from 22 suicide victims and 22 controls. Frontal cortex samples were diseected, frozen, and stored until assayed. Samples of tissue homogenate were incubated in duplicate with 10 concentrations of tritium-QNB. Specific binding was determined with and without atropine. The results confirmed previous studies in which no changes were noted in suicide versus control brains. While the findings neither disprove nor support the cholinergic hypothesis of depression, they do suggest that the neurochemical basis for the in vivo observations of increased responsivity of depressed individuals to muscarinic cholinergic agents might not involve changes in receptors estimated by QNB binding

  5. Selective Cancer Targeting via Aberrant Behavior of Cancer Cell-associated Glucocorticoid Receptor

    Mukherjee, Amarnath; Narayan, Kumar P; Pal, Krishnendu; Kumar, Jerald M.; Rangaraj, Nandini; Shasi V Kalivendi; Banerjee, Rajkumar

    2009-01-01

    Glucocorticoid receptors (GRs) are ubiquitous, nuclear hormone receptors residing in cell types of both cancer and noncancerous origin. It is not known whether cancer cell–associated GR alone can be selectively manipulated for delivery of exogenous genes to its nucleus for eliciting anticancer effect. We find that GR ligand, dexamethasone (Dex) in association with cationic lipoplex (termed as targeted lipoplex) could selectively manipulate GR in cancer cells alone for the delivery of transgen...

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

    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.)

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

    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.

  8. Malignant Peripheral Nerve Sheath Tumor Invasion Requires Aberrantly Expressed Epidermal Growth Factor (EGF) Receptors and is Variably Enhanced by Multiple EGF Family Ligands

    Byer, Stephanie J.; Brossier, Nicole M.; Peavler, Lafe T.; Eckert, Jenell M.; Watkins, Stacey; Roth, Kevin A.; Carroll, Steven L.

    2013-01-01

    Aberrant epidermal growth factor receptor (EGFR) expression promotes the pathogenesis of malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1, but the mechanisms by which EGFR expression promotes MPNST pathogenesis are poorly understood. We hypothesized that inappropriately expressed EGFRs promote MPNST invasion and found that these kinases are concentrated in MPNST invadopodia in vitro. EGFR knockdown inhibited the migration of unstimulated MPNST cells in vitro and exogenous EGF further enhanced MPNST migration in a substrate-specific manner, promoting migration on laminin and, to a lesser extent, collagen. Thus, in this setting, EGF acts as a chemotactic factor. We also found that the 7 known EGFR ligands (EGF, betacellulin, epiregulin, heparin-binding EGF, transforming growth factor α [TGFα], amphiregulin, and epigen) variably enhanced MPNST migration in a concentration-dependent manner, with TGFα being particularly potent. With the exception of epigen, these factors similarly promoted the migration of non-neoplastic Schwann cells. Although transcripts encoding all 7 EGFR ligands were detected in human MPNST cells and tumor tissues, only TGFα was consistently overexpressed and was found to colocalize with EGFR in situ. These data indicate that constitutive EGFR activation, potentially driven by autocrine or paracrine TGFα signaling, promotes the aggressive invasive behavior characteristic of MPNSTs. PMID:23399900

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

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

    2003-01-01

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

  10. Receptor binding characteristics and cytotoxicity of insulin-methotrexate

    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.

  11. Midbrain dopamine D2/3 receptor binding in schizophrenia.

    Tuppurainen, Heli; Kuikka, Jyrki T; Laakso, Mikko P; Viinamäki, Heimo; Husso, Minna; Tiihonen, Jari

    2006-09-01

    Several studies suggest that dysregulation of dopaminergic transmission in the midbrain and thalamus may contribute to the symptomatology of schizophrenia. The objective of this study was to examine the putative alteration of dopamine D(2/3 )receptor densities in the thalamus and midbrain of drug-naïve schizophrenic patients. We used the high-affinity single-photon emission tomography ligand [(123)I]epidepride for imaging D(2/3 )receptor binding sites in six neuroleptic-naïve schizophrenic patients, and seven healthy controls. Schizophrenic symptoms were evaluated by the Positive and Negative Syndrome Scale. Significantly lower D(2/3 )values were observed in the midbrain of patients with schizophrenia compared to controls (P = 0.02). No statistically significant difference was observed in the thalamus between two groups. Negative correlations were found between thalamic D(2/3 )receptor binding and general psychopathological schizophrenic symptoms (r from -0.78 to -0.92). These observations implicate altered dopaminergic activity in the midbrain of schizophrenic patients. PMID:16783502

  12. The glycocalyx promotes cooperative binding and clustering of adhesion receptors.

    Xu, Guang-Kui; Qian, Jin; Hu, Jinglei

    2016-05-18

    Cell adhesion plays a pivotal role in various biological processes, e.g., immune responses, cancer metastasis, and stem cell differentiation. The adhesion behaviors depend subtly on the binding kinetics of receptors and ligands restricted at the cell-substrate interfaces. Although much effort has been directed toward investigating the kinetics of adhesion molecules, the role of the glycocalyx, anchored on cell surfaces as an exterior layer, is still unclear. In this paper, we propose a theoretical approach to study the collective binding kinetics of a few and a large number of binders in the presence of the glycocalyx, representing the cases of initial and mature adhesions of cells, respectively. The analytical results are validated by finding good agreement with our Monte Carlo simulations. In the force loading case, the on-rate and affinity increase as more bonds form, whereas this cooperative effect is not observed in the displacement loading case. The increased thickness and stiffness of the glycocalyx tend to decrease the affinity for a few bonds, while they have less influence on the affinity for a large number of bonds. Moreover, for a flexible membrane with thermally-excited shape fluctuations, the glycocalyx is exhibited to promote the formation of bond clusters, mainly due to the cooperative binding of binders. This study helps to understand the cooperative kinetics of adhesion receptors under physiologically relevant loading conditions and sheds light on the novel role of the glycocalyx in cell adhesion. PMID:27102288

  13. Treponema pallidum receptor binding proteins interact with fibronectin

    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. Treponema pallidum receptor binding proteins interact with fibronectin

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

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

    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.

  16. 1918 Influenza receptor binding domain variants bind and replicate in primary human airway cells regardless of receptor specificity.

    Davis, A Sally; Chertow, Daniel S; Kindrachuk, Jason; Qi, Li; Schwartzman, Louis M; Suzich, Jon; Alsaaty, Sara; Logun, Carolea; Shelhamer, James H; Taubenberger, Jeffery K

    2016-06-01

    The 1918 influenza pandemic caused ~50 million deaths. Many questions remain regarding the origin, pathogenicity, and mechanisms of human adaptation of this virus. Avian-adapted influenza A viruses preferentially bind α2,3-linked sialic acids (Sia) while human-adapted viruses preferentially bind α2,6-linked Sia. A change in Sia preference from α2,3 to α2,6 is thought to be a requirement for human adaptation of avian influenza viruses. Autopsy data from 1918 cases, however, suggest that factors other than Sia preference played a role in viral binding and entry to human airway cells. Here, we evaluated binding and entry of five 1918 influenza receptor binding domain variants in a primary human airway cell model along with control avian and human influenza viruses. We observed that all five variants bound and entered cells efficiently and that Sia preference did not predict entry of influenza A virus to primary human airway cells evaluated in this model. PMID:27062579

  17. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

    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

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

    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

  19. Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia

    Lee-Sherick, A B; Eisenman, K M; Sather, S; McGranahan, A; Armistead, P M; McGary, C S; Hunsucker, S A; Schlegel, J.; Martinson, H; Cannon, C; Keating, A K; Earp, H S; Liang, X; DeRyckere, D; Graham, D K

    2013-01-01

    Acute myeloid leukemia (AML) continues to be extremely difficult to treat successfully, and the unacceptably low overall survival rates mandate that we assess new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation in AML has been associated with poor prognosis and provides strategic targets for novel therapy development. We found that Mer receptor tyrosine kinase was over-expressed in a majority of pediatric (29/36, 80%) and a...

  20. Outsmarting androgen receptor: creative approaches for targeting aberrant androgen signaling in advanced prostate cancer

    Karen E Knudsen; Kelly, William Kevin

    2011-01-01

    Prostatic adenocarcinomas are reliant on androgen receptor (AR) activity for survival and progression. Therefore, first-line therapeutic intervention for disseminated disease entails the use of AR-directed therapeutics, achieved through androgen deprivation and direct AR antagonists. While initially effective, recurrent, ‘castrate-resistant’ prostate cancers arise, for which there is no durable means of treatment. An abundance of clinical study and preclinical modeling has led to the revelati...

  1. Binding affinity prediction of novel estrogen receptor ligands using receptor-based 3-D QSAR methods.

    Sippl, Wolfgang

    2002-12-01

    We have recently reported the development of a 3-D QSAR model for estrogen receptor ligands showing a significant correlation between calculated molecular interaction fields and experimentally measured binding affinity. The ligand alignment obtained from docking simulations was taken as basis for a comparative field analysis applying the GRID/GOLPE program. Using the interaction field derived with a water probe and applying the smart region definition (SRD) variable selection procedure, a significant and robust model was obtained (q(2)(LOO)=0.921, SDEP=0.345). To further analyze the robustness and the predictivity of the established model several recently developed estrogen receptor ligands were selected as external test set. An excellent agreement between predicted and experimental binding data was obtained indicated by an external SDEP of 0.531. Two other traditionally used prediction techniques were applied in order to check the performance of the receptor-based 3-D QSAR procedure. The interaction energies calculated on the basis of receptor-ligand complexes were correlated with experimentally observed affinities. Also ligand-based 3-D QSAR models were generated using program FlexS. The interaction energy-based model, as well as the ligand-based 3-D QSAR models yielded models with lower predictivity. The comparison with the interaction energy-based model and with the ligand-based 3-D QSAR models, respectively, indicates that the combination of receptor-based and 3-D QSAR methods is able to improve the quality of prediction. PMID:12413831

  2. Localization of CGRP receptor components, CGRP, and receptor binding sites in human and rhesus cerebellar cortex

    Eftekhari, Sajedeh; Salvatore, Christopher A; Gaspar, Renee C;

    2013-01-01

    receptor activity modifying protein 1 (RAMP1), was examined. In addition, expression of procalcitonin was studied. We observed high [(3)H]MK-3207 (CGRP receptor antagonist) binding densities in the molecular layer of rhesus cerebellar cortex; however, due to the limit of resolution of the autoradiographic....... Immunofluorescence revealed expression of CGRP, CLR, and RAMP1 in the Purkinje cells and in cells in the molecular layer. Procalcitonin was found in the same localization. Recent research in the biology of cerebellum indicates that it may have a role in nociception. For the first time we have identified CGRP and...

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

    Licht, Cecilie Löe; 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....... 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...

  4. Differential estrogen receptor binding of estrogenic substances: a species comparison.

    Matthews, J; Celius, T; Halgren, R; Zacharewski, T

    2000-11-15

    The study investigated the ability of 34 natural and synthetic chemicals to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed glutathione-S-transferase (GST)-estrogen receptors (ER) fusion proteins from five different species. Fusion proteins consisted of the ER D, E and F domains of human alpha (GST-hERalphadef), mouse alpha (GST-mERalphadef), chicken (GST-cERdef), green anole (GST-aERdef) and rainbow trout ERs (GST-rtERdef). All five fusion proteins displayed high affinity for E2 with dissociation constants (K(d)) ranging from 0.3 to 0.9 nM. Although, the fusion proteins exhibited similar binding preferences and binding affinities for many of the chemicals, several differences were observed. For example, alpha-zearalenol bound with greater affinity to GST-rtERdef than E2, which was in contrast to other GST-ERdef fusion proteins examined. Coumestrol, genistein and naringenin bound with higher affinity to the GST-aERdef, than to the other GST-ERdef fusion proteins. Many of the industrial chemicals examined preferentially bound to GST-rtERdef. Bisphenol A, 4-t-octylphenol and o,p' DDT bound with approximately a ten-fold greater affinity to GST-rtERdef than to other GST-ERdefs. Methoxychlor, p,p'-DDT, o,p'-DDE, p,p'-DDE, alpha-endosulfan and dieldrin weakly bound to the ERs from the human, mouse, chicken and green anole. In contrast, these compounds completely displaced [3H]E2 from GST-rtERdef. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for estrogenic compounds and that these differences may be due to the variability in the amino acid sequence within their respective ER ligand binding domains. PMID:11162928

  5. The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

    Bonde, Marie Mi; Olsen, Kristine Boisen; Erikstrup, Niels;

    2011-01-01

    The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately...... connected and some of the cardioprotective effects of Losartan are abolished by blocking the bradykinin B2 receptor (B2R) signaling. In this study, we investigated the ability of six clinically available ARBs to specifically bind and activate the B2R. First, we investigated their ability to activate...... phosphoinositide (PI) hydrolysis in COS-7 cells transiently expressing the B2R. We found that only Losartan activated the B2R, working as a partial agonist compared to the endogenous ligand bradykinin. This effect was blocked by the B2R antagonist HOE 140. A competitive binding analysis revealed that Losartan does...

  6. Benzodiazepine receptor binding in vivo with (/sup 3/)-Ro 15-1788

    Goeders, N.E.; Kuhar, M.J.

    1985-07-29

    In vivo benzodiazepine receptor binding has generally been studied by ex vivo techniques. In this investigation, the authors identify the conditions where (/sup 3/H)-Ro 15-1788 labels benzodiazepine receptors by true in vivo binding, i.e. where workable specific to nonspecific ratios are obtained in intact tissues without homogenization or washing. (/sup 3/H)-Flunitrazepam and (/sup 3/H)-clonazepam did not exhibit useful in vivo receptor binding. 39 references, 5 figures, 1 table.

  7. Benzodiazepine receptor binding in vivo with [3]-Ro 15-1788

    In vivo benzodiazepine receptor binding has generally been studied by ex vivo techniques. In this investigation, the authors identify the conditions where [3H]-Ro 15-1788 labels benzodiazepine receptors by true in vivo binding, i.e. where workable specific to nonspecific ratios are obtained in intact tissues without homogenization or washing. [3H]-Flunitrazepam and [3H]-clonazepam did not exhibit useful in vivo receptor binding. 39 references, 5 figures, 1 table

  8. Identification of Essential Cannabinoid-binding Domains: STRUCTURAL INSIGHTS INTO EARLY DYNAMIC EVENTS IN RECEPTOR ACTIVATION*

    Shim, Joong-Youn; Bertalovitz, Alexander C.; Kendall, Debra A.

    2011-01-01

    The classical cannabinoid agonist HU210, a structural analog of (−)-Δ9-tetrahydrocannabinol, binds to brain cannabinoid (CB1) receptors and activates signal transduction pathways. To date, an exact molecular description of the CB1 receptor is not yet available. Utilizing the minor binding pocket of the CB1 receptor as the primary ligand interaction site, we explored HU210 binding using lipid bilayer molecular dynamics (MD) simulations. Among the potential ligand contact residues, we identifie...

  9. Influences of hydrocarbon linkers on the receptor binding affinities of gonadotropin-releasing hormone peptides

    Guo, Haixun; Hathaway, Helen; Royce, Melanie E.; Prossnitz, Eric R.; Miao, Yubin

    2013-01-01

    Three new DOTA-conjugated GnRH peptides with various hydrocarbon linkers were synthesized to evaluate the influences of the linkers on their receptor binding affinities. The hydrocarbon linker displayed a profound impact on the receptor binding affinities of DOTA-conjugated GnRH peptides. The Aun linker was better than Gaba, Ahx and Aoc linkers in retaining strong receptor binding affinity of the GnRH peptide. DOTA-Aun-(D-Lys6-GnRH) displayed 22.8 nM GnRH receptor binding affinity. 111In-DOTA...

  10. Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

    The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increased ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids

  11. Complement Component C3 Binds to Activated Normal Platelets without Preceding Proteolytic Activation and Promotes Binding to Complement Receptor 1

    Osama A Hamad; Nilsson, Per H.; Wouters, Diana; Lambris, John D.; Ekdahl, Kristina N.; Nilsson, Bo

    2010-01-01

    It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfaces of nonactivated and thrombin receptor-activating peptide-activated platelets was analyzed by flow cytometry and Western blot analyses. C1q, C4, C3, and C9 were found to bind to thrombin receptor...

  12. Aberrant methylation of the M-type phospholipase A2 receptor gene in leukemic cells

    The M-type phospholipase A2 receptor (PLA2R1) plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS) or acute leukemia. Sites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM) analysis was then carried out to quantify PLA2R1 methylation at 5-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA. Expression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2´-deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% ± 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia. The study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis

  13. Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia.

    Lee-Sherick, A B; Eisenman, K M; Sather, S; McGranahan, A; Armistead, P M; McGary, C S; Hunsucker, S A; Schlegel, J; Martinson, H; Cannon, C; Keating, A K; Earp, H S; Liang, X; DeRyckere, D; Graham, D K

    2013-11-14

    Acute myeloid leukemia (AML) continues to be extremely difficult to treat successfully, and the unacceptably low overall survival rates mandate that we assess new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation in AML has been associated with poor prognosis and provides strategic targets for novel therapy development. We found that Mer receptor tyrosine kinase was over-expressed in a majority of pediatric (29/36, 80%) and adult (10/10, 100%) primary AML patient blasts at the time of diagnosis, and 100% of patient samples at the time of relapse. Mer was also found to be expressed in 12 of 14 AML cell lines (86%). In contrast, normal bone marrow myeloid precursors expressed little to no Mer. Following AML cell line stimulation with Gas6, a Mer ligand, we observed activation of prosurvival and proliferative signaling pathways, including phosphorylation of ERK1/2, p38, MSK1, CREB, ATF1, AKT and STAT6. To assess the phenotypic role of Mer in AML, two independent short-hairpin RNA (shRNA) constructs were used to decrease Mer expression in the AML cell lines Nomo-1 and Kasumi-1. Reduction of Mer protein levels significantly increased rates of myeloblast apoptosis two to threefold in response to serum starvation. Furthermore, myeloblasts with knocked-down Mer demonstrated decreased colony formation by 67-87%, relative to control cell lines (P<0.01). NOD-SCID-gamma mice transplanted with Nomo-1 myeloblasts with reduced levels of Mer had a significant prolongation in survival compared with mice transplanted with the parental or control cell lines (median survival 17 days in parental and control cell lines, versus 32-36 days in Mer knockdown cell lines, P<0.0001). These data suggest a role for Mer in acute myeloid leukemogenesis and indicate that targeted inhibition of Mer may be an effective therapeutic strategy in pediatric and adult AML. PMID:23474756

  14. Aberrant methylation of the M-type phospholipase A2 receptor gene in leukemic cells

    Menschikowski Mario

    2012-12-01

    Full Text Available Abstract Background The M-type phospholipase A2 receptor (PLA2R1 plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS or acute leukemia. Methods Sites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM analysis was then carried out to quantify PLA2R1 methylation at 5`-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA. Results Expression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2´-deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% ± 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia. Conclusions The study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis.

  15. Aberrant Activation of the RANK Signaling Receptor Induces Murine Salivary Gland Tumors.

    Maria M Szwarc

    Full Text Available Unlike cancers of related exocrine tissues such as the mammary and prostate gland, diagnosis and treatment of aggressive salivary gland malignancies have not markedly advanced in decades. Effective clinical management of malignant salivary gland cancers is undercut by our limited knowledge concerning the key molecular signals that underpin the etiopathogenesis of this rare and heterogeneous head and neck cancer. Without knowledge of the critical signals that drive salivary gland tumorigenesis, tumor vulnerabilities cannot be exploited that allow for targeted molecular therapies. This knowledge insufficiency is further exacerbated by a paucity of preclinical mouse models (as compared to other cancer fields with which to both study salivary gland pathobiology and test novel intervention strategies. Using a mouse transgenic approach, we demonstrate that deregulation of the Receptor Activator of NFkB Ligand (RANKL/RANK signaling axis results in rapid tumor development in all three major salivary glands. In line with its established role in other exocrine gland cancers (i.e., breast cancer, the RANKL/RANK signaling axis elicits an aggressive salivary gland tumor phenotype both at the histologic and molecular level. Despite the ability of this cytokine signaling axis to drive advanced stage disease within a short latency period, early blockade of RANKL/RANK signaling markedly attenuates the development of malignant salivary gland neoplasms. Together, our findings have uncovered a tumorigenic role for RANKL/RANK in the salivary gland and suggest that targeting this pathway may represent a novel therapeutic intervention approach in the prevention and/or treatment of this understudied head and neck cancer.

  16. An aprotinin binding site localized in the hormone binding domain of the estrogen receptor from calf uterus.

    Nigro, V; Medici, N; Abbondanza, C; Minucci, S; Moncharmont, B; Molinari, A M; Puca, G A

    1990-07-31

    It has been proposed that the estrogen receptor bears proteolytic activity responsible for its own transformation. This activity was inhibited by aprotinin. Incubation of transformed ER with aprotinin modified the proteolytic digestion of the hormone binding subunit by proteinase K. The smallest hormone-binding fragment of the ER, obtained by tryptic digestion, was still able to bind to aprotinin. These results suggest that aprotinin interacts with ER and the hormone-binding domain of ER is endowed with a specific aprotinin-binding site. PMID:1696480

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

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

    2010-01-01

    For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low......-density lipoprotein receptor-related protein 1 (LRP1). We show here that on sensory neurons LRP1 is also the receptor that binds and rapidly endocytoses smaller oligomeric forms of infectious prion fibrils, and recombinant PrP fibrils. Although LRP1 binds two molecules of most ligands independently to its receptor...... both prion and LRP1 biology....

  18. Mapping convulsants’ binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites

    Kalueff, A.V.

    2006-01-01

    Gamma aminobutyric acid (GABA) type A receptors play a key role in brain inhibitory neurotransmission, and are ligand-activated chloride channels blocked by numerous convulsant ligands. Here we summarize data on binding of picrotoxin, tetrazoles, β-lactams, bicyclophosphates, butyrolactones and neurotoxic pesticides to GABA-A ionophore, and discuss functional and structural overlapping of their binding sites. The paper reviews data on convulsants’ binding sensitivity to different point mutati...

  19. Mutation in apolipoprotein B associated with hypobetalipoproteinemia despite decreased binding to the low density lipoprotein receptor

    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...

  20. Identification of essential cannabinoid-binding domains: structural insights into early dynamic events in receptor activation.

    Shim, Joong-Youn; Bertalovitz, Alexander C; Kendall, Debra A

    2011-09-23

    The classical cannabinoid agonist HU210, a structural analog of (-)-Δ(9)-tetrahydrocannabinol, binds to brain cannabinoid (CB1) receptors and activates signal transduction pathways. To date, an exact molecular description of the CB1 receptor is not yet available. Utilizing the minor binding pocket of the CB1 receptor as the primary ligand interaction site, we explored HU210 binding using lipid bilayer molecular dynamics (MD) simulations. Among the potential ligand contact residues, we identified residues Phe-174(2.61), Phe-177(2.64), Leu-193(3.29), and Met-363(6.55) as being critical for HU210 binding by mutational analysis. Using these residues to guide the simulations, we determined essential cannabinoid-binding domains in the CB1 receptor, including the highly sought after hydrophobic pocket important for the binding of the C3 alkyl chain of classical and nonclassical cannabinoids. Analyzing the simulations of the HU210-CB1 receptor complex, the CP55940-CB1 receptor complex, and the (-)-Δ(9)-tetrahydrocannabinol-CB1 receptor complex, we found that the positioning of the C3 alkyl chain and the aromatic stacking between Trp-356(6.48) and Trp-279(5.43) is crucial for the Trp-356(6.48) rotamer change toward receptor activation through the rigid-body movement of H6. The functional data for the mutant receptors demonstrated reductions in potency for G protein activation similar to the reductions seen in ligand binding affinity for HU210. PMID:21795705

  1. Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding

    Previous in vivo turnover studies suggested that retarded clearance of low density lipoproteins (LDL) from the plasma of some hypercholesterolemic patients is due to LDL with defective receptor binding. The present study examined this postulate directly by receptor binding experiments. The LDL from a hypercholesterolemic patient (G.R.) displayed a reduced ability to bind to the LDL receptors on normal human fibroblasts. The G.R. LDL possessed 32% of normal receptor binding activity. Likewise, the G.R. LDL were much less effective than normal LDL in competing with 125I-labeled normal LDL for cellular uptake and degradation and in stimulating intracellular cholesteryl ester synthesis. The defect in LDL binding appears to be due to a genetic abnormality of apolipoprotein B-100: two brothers of the proband possess LDL defective in receptor binding, whereas a third brother and the proband's son have normally binding LDL. Further, the defect in receptor binding does not appear to be associated wit an abnormal lipid composition or structure of the LDL. Normal and abnormal LDL subpopulations were partially separated from plasma of two subjects by density-gradient ultracentrifugation, a finding consistent with the presence of a normal and a mutant allele. The affected family members appear to be heterozygous for this disorder, which has been designated familial defective apolipoprotein B-100. These studies indicate that the defective receptor binding results in inefficient clearance of LDL and the hypercholesterolemia observed in these patients

  2. Substance P receptor binding sites are expressed by glia in vivo after neuronal injury

    In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, the authors examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system

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

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

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

  4. Competitive inhibition of [3H]dexamethasone binding to mammary glucocorticoid receptor by leupeptin

    The inhibitory effect of leupeptin on [3H]dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of [3H]dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of [3H]dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S)

  5. Aberrant methylation of N-methyl-D-aspartate receptor type 2B (NMDAR2B) in non-small cell carcinoma

    N-methyl-D-aspartate receptors (NMDAR) act as tumor suppressors of digestive malignancies. The expression and genetic methylation patterns of NMDAR2B in non-small cell lung cancer (NSCLC) are unknown. The relationship between gene methylation and expression of NMDAR2B was analyzed in NSCLC cell lines (N = 9) and clinical tissues (N = 216). The cell lines were studied using RT-PCR and 5-aza-2'-deoxycytidine treatment, while the clinical tissues were examined by methylation specific real-time quantitative PCR and immunohistochemistry. Retrospective investigation of patient records was used to determine the clinical significance of NMDAR2B methylation. NMDAR2B was silenced in five of the nine cell lines; 5-aza-2'-deoxycytidine treatment restored expression, and was inversely correlated with methylation. Aberrant methylation of NMDAR2B, detected in 61% (131/216) of clinical NSCLC tissues, was inversely correlated with the status of protein expression in 20 randomly examined tumors. Aberrant methylation was not associated with clinical factors such as gender, age, histological type, or TNM stage. However, aberrant methylation was an independent prognostic factor in squamous cell carcinoma cases. Aberrant methylation of the NMDAR2B gene is a common event in NSCLC. The prognosis was significantly better for cases of squamous cell carcinoma in which NMDAR2B was methylated. It may have different roles in different histological types

  6. Metal binding sites of the estradiol receptor from calf uterus and their possible role in the regulation of receptor function

    The existence of putative metal binding sites on the estradiol receptor (ER) molecule from calf uterus was evaluated by immobilizing various divalent metals to iminodiacetate-Sepharose. ER from both crude and highly purified preparations binds to metal-containing adsorbents complexed with Zn(II), Ni(II), Co(II), and Cu(II), but not to those complexed with Fe(II) and Cd(II). Analysis of affinity-labeled ER by [3H]tamoxifen aziridine after elution from a column of Zn(II)-charged iminodiacetate-Sepharose showed that ER fragments obtained by extensive trypsinization were also bound. Zn(II) and the same other metals able to bind ER, when immobilized on resins, inhibit the binding of estradiol to the receptor at micromolar concentration. This inhibition is noncompetitive and can be reversed by EDTA. The inhibition of the hormone binding was still present after trypsin treatment of the cytosol, and it was abolished by preincubation with the hormone. Micromolar concentrations of these metals were able to block those chemical-physical changes occurring during the process of ER transformation in vitro. The presence of metal binding sites that modulate the ER activity in the hormone binding domain of ER is speculated. Since progesterone receptor showed the same pattern of binding and elution from metal-containing adsorbents, the presence of metal binding regulatory sites could be a property of all steroid receptors

  7. Metal binding sites of the estradiol receptor from calf uterus and their possible role in the regulation of receptor function

    Medici, N.; Minucci, S.; Nigro, V.; Abbondanza, C.; Armetta, I.; Molinari, A.M.; Puca, G.A. (Istituto di Patologia Generale ed Oncologia, Naples (Italy))

    1989-01-10

    The existence of putative metal binding sites on the estradiol receptor (ER) molecule from calf uterus was evaluated by immobilizing various divalent metals to iminodiacetate-Sepharose. ER from both crude and highly purified preparations binds to metal-containing adsorbents complexed with Zn(II), Ni(II), Co(II), and Cu(II), but not to those complexed with Fe(II) and Cd(II). Analysis of affinity-labeled ER by ({sup 3}H)tamoxifen aziridine after elution from a column of Zn(II)-charged iminodiacetate-Sepharose showed that ER fragments obtained by extensive trypsinization were also bound. Zn(II) and the same other metals able to bind ER, when immobilized on resins, inhibit the binding of estradiol to the receptor at micromolar concentration. This inhibition is noncompetitive and can be reversed by EDTA. The inhibition of the hormone binding was still present after trypsin treatment of the cytosol, and it was abolished by preincubation with the hormone. Micromolar concentrations of these metals were able to block those chemical-physical changes occurring during the process of ER transformation in vitro. The presence of metal binding sites that modulate the ER activity in the hormone binding domain of ER is speculated. Since progesterone receptor showed the same pattern of binding and elution from metal-containing adsorbents, the presence of metal binding regulatory sites could be a property of all steroid receptors.

  8. Aprotinin inhibits the hormone binding of the estrogen receptor from calf uterus.

    Nigro, V; Medici, N; Abbondanza, C; Minucci, S; Molinari, A M; Puca, G A

    1989-11-15

    Micromolar concentrations of the proteinase inhibitor, aprotinin, produced a dose-dependent inhibition in the binding capacity of the estrogen receptor from calf uterus. Aprotinin inhibition was greater at 28 degrees C than at 4 degrees C and only occurred when conditions allowed the receptor transformation. When aprotinin was tested in the presence of transformation inhibitors, its effect was no longer seen. The binding capacity of the highly purified estrogen-binding subunit was similarly inhibited. PMID:2480113

  9. [3H]Ethylketocyclazocine binding to mouse brain membranes: evidence for a kappa opioid receptor type

    The binding of the putative kappa agonist ethylketocyclazocine (EKC) to synaptosomal membranes of mouse brain was studied. This benzomorphan was able to bind to different opioid receptors. A portion of this binding was not inhibited by the agonist naloxone, even at high concentrations (10 microM). This population of receptors, to which opioate alkaloids and opiod peptides display very low affinity, is probably the sigma receptor. Another class of binding sites was identified by the simultaneous addition of the selective agonists Sandoz FK-33824 and D-Ala2-D-Leu5-enkephalin, which blocked the access of EKC to mu and delta opioid receptors, respectively, leaving a portion of naloxone-displaceable benzomorphan binding still detectable. Analysis of this remaining binding revealed a small population of receptors of high affinity, the kappa receptor. Therefore, EKC binds to the mu, delta, kappa and sigma receptors in the mouse brain, with similar affinities for the mu and kappa (0.22 and 0.15 nM). These results confirm the existence of a kappa opioid receptor type in the mouse brain

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

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

    2009-01-01

    currently available crystal structures. This was used to characterize pharmacological relationships of Family A/Rhodopsin family GPCRs, minimizing evolutionary influence from parts of the receptor that do not generally affect ligand binding. The resultant dendogram tended to group receptors according to...

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

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

    2010-01-01

    The 5-HT(4) receptor is a new potential target for antidepressant treatment and may be implicated in the pathogenesis of depression. This study investigated differences in 5-HT(4) receptor and 5-HT transporter (5-HTT) binding by quantitative autoradiography of [(3)H]SB207145 and (S)-[N-methyl-(3)...

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

    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...

  13. The human enhancer-binding protein Gata3 binds to several T-cell receptor regulatory elements.

    Marine, J; Winoto, A

    1991-01-01

    The tissue-specific developmental regulation of the alpha, beta, gamma and delta T-cell antigen receptor (TCR) genes is controlled by the corresponding distinct enhancers and their enhancer-binding proteins. To find a common TCR regulatory element, we have studied the ability of the newly described enhancer-binding protein Gata3 to bind to the sequence motif (A/T)GATA(G/A) shared between enhancer elements of all four TCR genes. Gata3 was shown in the chicken to be an enhancer-binding protein ...

  14. Neurotensin receptor binding levels in basal ganglia are not altered in Huntington's chorea or schizophrenia

    Autoradiographic techniques were used to examine the distribution and levels of neurotensin receptor binding sites in the basal ganglia and related regions of the human brain. Monoiodo (125I-Tyr3)neurotensin was used as a ligand. High amounts of neurotensin receptor binding sites were found in the substantia nigra pars compacta. Lower but significant quantities of neurotensin receptor binding sites characterized the caudate, putamen, and nucleus accumbens, while very low quantities were seen in both medial and lateral segments of the globus pallidus. In Huntington's chorea, the levels of neurotensin receptor binding sites were found to be comparable to those of control cases. Only slight but not statistically significant decreases in amounts of receptor binding sites were detected in the dorsal part of the head and in the body of caudate nucleus. No alterations in the levels of neurotensin receptor binding sites were observed in the substantia nigra pars compacta and reticulata. These results suggest that a large proportion of neurotensin receptor binding sites in the basal ganglia are located on intrinsic neurons and on extrinsic afferent fibers that do not degenerate in Huntington's disease

  15. Effects of vitamin B-6 nutrition on benzodiazepine (BDZ) receptor binding in the developing rat brain

    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 3H-flunitrazepam as ligand were performed in the presence and absence of 10 μM 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 3H-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

  16. Chromosomal aberration

    Chromosomal aberrations are classified into two types, chromosome-type and chromatid-type. Chromosom-type aberrations include terminal deletion, dicentric, ring and interstitial deletion, and chromatid-type aberrations include achromatic lesion, chromatid deletion, isochromatid deletion and chromatid exchange. Clastogens which induce chromosomal aberration are divided into ''S-dependent'' agents and ''S-independent''. It might mean whether they can induce double strand breaks independent of the S phase or not. Double strand breaks may be the ultimate lesions to induce chromosomal aberrations. Caffeine added even in the G2 phase appeared to modify the frequency of chromatid aberrations induced by X-rays and mitomycin C. Those might suggest that the G2 phase involves in the chromatid aberration formation. The double strand breaks might be repaired by ''G2 repair system'', the error of which might yield breakage types of chromatid aberrations and the by-pass of which might yield chromatid exchanges. Chromosome-type aberrations might be formed in the G1 phase. (author)

  17. Calcitonin receptor binding properties in bone and kidney of the chicken during the oviposition cycle.

    Yasuoka, T; Kawashima, M; Takahashi, T; Tatematsu, N; Tanaka, K

    1998-09-01

    The binding property of calcitonin (CT) in the membrane fraction of calvaria and kidney of egg-laying and nonlaying hens was analyzed using a [125I] CT binding assay system. Binding properties of CT receptors in both tissues satisfy the authentic criteria of a receptor-ligand interaction in terms of specificity, reversibility, and saturation. Scatchard plots revealed a single class of binding sites. Values of the equilibrium dissociation constant (Kd) and binding capacity (Bmax) in laying hens showed a decrease during the period between 3 h before and 2 h after oviposition. No change was observed in nonlaying hens. In vivo administration of 17beta-estradiol or progesterone caused the decrease in Kd and Bmax values. The results suggest that the binding affinity and capacity of the CT receptor in the calvaria and the kidney of the hen may be modulated by the ovarian steroid hormone. PMID:9738513

  18. Human myometrial adrenergic receptors: identification of the beta-adrenergic receptor by [3H]dihydroalprenolol binding

    The radioactive beta-adrenergic antagonist [3H] dihydroalprenolol (DHA) binds to particulate preparations of human myometrium in a manner compatible with binding to the beta-adrenergic receptor. The binding of DHA is rapid (attaining equilibrium in 12 minutes), readily reversible (half time = 16 minutes), high affinity (K/sub D/ = 0.50 nM), low capacity (Bmax = 70 fmoles/mg of protein), and stereoselective ([-]-propranolol is 100 times as potent as [+] -propranolol in inhibiting DHA binding). Adrenergic agonists competed for DHA binding sites in a manner compatible with beta-adrenergic interactions and mirrored β2 pharmacologic potencies: isoproterenol > epinephrine >> norepinephrine. Studies in which zinterol, a β2-adrenergic agonist, competed for DHA binding sites in human myometrial particulate indicated that at least 87% of the beta-adrenergic receptors present are β2-adrenergic receptors. Binding of DHA to human myometrial beta-adrenergic receptors provides a tool which may be used in the examination of gonadal hormonal modification of adrenergic response in human uterus as well as in the analysis of beta-adrenergic agents as potentially useful tocolytic agents

  19. Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo

    Shibusawa, Nobuyuki; Hashimoto, Koshi; Nikrodhanond, Amisra A.; Liberman, M. Charles; Applebury, Meredithe L.; Liao, Xiao Hui; Robbins, Janet T.; Refetoff, Samuel; Cohen, Ronald N.; Wondisford, Fredric E.

    2003-01-01

    Thyroid hormone action is mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. DNA-binding is presumed to be essential for all nuclear actions of thyroid hormone. To test this hypothesis in vivo, the DNA-binding domain of TR-β was mutated within its P-box (GS mutant) using gene targeting techniques. This mutation in vitro completely abolishes TR-β DNA-binding, while preserving ligand (T3) and cofactor interactions with the receptor. Homoz...

  20. Binding ability of LHRH-PE40 to LHRH receptors on cancer cell line

    Objective: To evaluate the binding ability of LHRH-PE40, a fusion protein, to the LHRH receptors on cancer cell line. Methods: The radioligand binding assay of receptors was used to calculate the Kd and Bmax. Results: Hela cell line: Kd=(0.36 +- 0.12) nmol, Bmax=(0.23+-0.15) μmol·g-1; Hep2 cell line: Kd=(0.33 +- 0.11) nmol, Bmax=(0.46 +- 0.12)μmol·g-1. Conclusion: LHRH-PE40 has a high binding affinity to the LHRH receptors on cancer cell line, which is the same as the natural LHRH

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

    Daughaday, W H; Trivedi, B

    1987-01-01

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

  2. Origin and evolution of the ligand-binding ability of nuclear receptors.

    Markov, Gabriel V; Laudet, Vincent

    2011-03-01

    The origin of the ligand-binding ability of nuclear receptors is still a matter of discussion. Current opposing models are the early evolution of an ancestral receptor that would bind a specific ligand with high affinity and the early evolution of an ancestral orphan that was a constitutive transcription factor. Here we review the arguments in favour or against these two hypotheses, and we discuss an alternative possibility that the ancestor was a ligand sensor, which would be able to explain the apparently contradictory data generated in previous models for the evolution of ligand binding in nuclear receptors. PMID:21055443

  3. Ligand Binding Sensitivity of the Extracellular Loop Two of the Cannabinoid Receptor 1

    Bertalovitz, Alexander C.; Ahn, Kwang H.; Kendall, Debra A.

    2010-01-01

    The cannabinoid receptor one (CB1) is a class A G-protein-coupled receptor thought to bind ligands primarily within its helical bundle. Evidence suggests, however, that the extracellular domain may also play a role. We have previously shown that the C-terminus of the extracellular loop 2 of CB1 is important in binding some compounds; receptors with mutations in this region (F268W, P269A, H270A, and I271A) bound some agonists with severely reduced affinity relative to the wild-type receptor. I...

  4. Ligand Binding Pathways of Clozapine and Haloperidol in the Dopamine D2 and D3 Receptors.

    Thomas, Trayder; Fang, Yu; Yuriev, Elizabeth; Chalmers, David K

    2016-02-22

    The binding of a small molecule ligand to its protein target is most often characterized by binding affinity and is typically viewed as an on/off switch. The more complex reality is that binding involves the ligand passing through a series of intermediate states between the solution phase and the fully bound pose. We have performed a set of 29 unbiased molecular dynamics simulations to model the binding pathways of the dopamine receptor antagonists clozapine and haloperidol binding to the D2 and D3 dopamine receptors. Through these simulations we have captured the binding pathways of clozapine and haloperidol from the extracellular vestibule to the orthosteric binding site and thereby, we also predict the bound pose of each ligand. These are the first long time scale simulations of haloperidol or clozapine binding to dopamine receptors. From these simulations, we have identified several important stages in the binding pathway, including the involvement of Tyr7.35 in a "handover" mechanism that transfers the ligand between the extracellular vestibule and Asp3.32. We have also performed interaction and cluster analyses to determine differences in binding pathways between the D2 and D3 receptors and identified metastable states that may be of use in drug design. PMID:26690887

  5. Binding domains of stimulatory and inhibitory thyrotropin (TSH) receptor autoantibodies determined with chimeric TSH-lutropin/chorionic gonadotropin receptors.

    Nagayama, Y; Wadsworth, H L; Russo, D.; Chazenbalk, G D; Rapoport, B

    1991-01-01

    We examined the relative effects of thyrotropin (TSH) and TSH receptor autoantibodies in the sera of patients with autoimmune thyroid disease on three TSH-lutropin/chorionic gonadotropin (LH/CG) receptor extracellular domain chimeras. Each chimera binds TSH with high affinity. Only the chimera with TSH receptor extracellular domains ABC (amino acids 1-260) had a functional (cAMP) response to thyroid stimulatory IgG. The chimeras with TSH receptor domains CD (amino acids 171-360) and DE (amino...

  6. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG3k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [3H]naloxone. The antibody which did not inhibit the binding of [3H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG3k antibody that blocked the binding of [3H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

  7. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    Conroy, W.G.

    1988-01-01

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG{sub 3}k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of ({sup 3}H)naloxone. The antibody which did not inhibit the binding of ({sup 3}H)naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG{sub 3}k antibody that blocked the binding of ({sup 3}H)naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form.

  8. Analyzing machupo virus-receptor binding by molecular dynamics simulations

    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. Analyzing machupo virus-receptor binding by molecular dynamics simulations.

    Meyer, Austin G; Sawyer, Sara L; Ellington, Andrew D; Wilke, Claus O

    2014-01-01

    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. PMID:24624315

  10. Metal binding sites of the estradiol receptor from calf uterus and their possible role in the regulation of receptor function.

    Medici, N; Minucci, S; Nigro, V; Abbondanza, C; Armetta, I; Molinari, A M; Puca, G A

    1989-01-10

    The existence of putative metal binding sites on the estradiol receptor (ER) molecule from calf uterus was evaluated by immobilizing various divalent metals to iminodiacetate-Sepharose. ER from both crude and highly purified preparations binds to metal-containing adsorbents complexed with Zn(II), Ni(II), Co(II), and Cu(II), but not to those complexed with Fe(II) and Cd(II). Elution of ER was obtained by chelating agents or by imidazole, thus indicating that histidine residues on the ER molecule are involved in the interaction with the metal. Analysis of affinity-labeled ER by [3H]tamoxifen aziridine after elution from a column of Zn(II)-charged iminodiacetate-Sepharose showed that ER fragments obtained by extensive trypsinization were also bound. Zn(II) and the same other metals able to bind ER, when immobilized on resins, inhibit the binding of estradiol to the receptor at micromolar concentrations. This inhibition is noncompetitive and can be reversed by EDTA. The inhibition of the hormone binding was still present after trypsin treatment of the cytosol, and it was abolished by preincubation with the hormone. Micromolar concentrations of these metals were able to block those chemical-physical changes occurring during the process of ER transformation in vitro. Furthermore, if added to pretransformed ER-hormone complex, they strongly inhibited the binding of the complex to isolated nuclei. The presence of metal binding sites that modulate the ER activity in the hormone binding domain of ER is therefore speculated. Since progesterone receptor showed the same pattern of binding and elution from metal-containing adsorbents, the presence of metal binding regulatory sites could be a property of all steroid receptors. PMID:2706244

  11. Specific receptor binding of staphylococcal enterotoxins by murine splenic lymphocytes.

    Buxser, S; Bonventre, P F; Archer, D L

    1981-01-01

    We describe a reliable assay to measure the specific binding of 125I-labeled staphylococcal enterotoxin A (SEA) by murine spleen cells. Toxin binding by lymphocytes was specific in that it was inhibited by unlabeled SEA but not by unrelated proteins. The biological activity of SEA (T-lymphocyte mitogenesis) correlated with toxin binding to splenic lymphocytes. In the presence of high concentrations of [125I]SEA, specific binding increased rapidly and approached saturation after 2 h. Toxin bin...

  12. Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography

    Gates, T.S.; Zimmerman, R.P.; Mantyh, C.R.; Vigna, S.R.; Maggio, J.E.; Welton, M.L.; Passaro, E.P. Jr.; Mantyh, P.W.

    1988-11-01

    Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for /sup 125/I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: (1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; (2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; (3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and (4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells.

  13. Mutation in apolipoprotein B associated with hypobetalipoproteinemia despite decreased binding to the low density lipoprotein receptor

    Benn, Marianne; Nordestgaard, Børge G; Jensen, Jan Skov; Nilausen, Karin Skov; Meinertz, Hans; Tybjaerg-Hansen, Anne

    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...... in vitro studies with both human in vivo and population-based studies, as in vitro studies often have focused on very limited aspects of complex mechanisms taken out of their natural context....

  14. Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy

    Jakubík, J; Janíčková, H; El-Fakahany, EE; Doležal, V

    2011-01-01

    BACKGROUND AND PURPOSE Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5′-γ−thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M2 muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH Filtration and scintillation proximity ass...

  15. Ligand binding and micro-switches in 7TM receptor structures

    Nygaard, Rie; Frimurer, Thomas M; Holst, Birgitte; Rosenkilde, Mette M; Schwartz, Thue W

    2009-01-01

    The past couple of years have seen several novel X-ray structures of 7 transmembrane (7TM) receptors in complex with antagonists and even with a peptide fragment of a G protein. These structures demonstrate that the main ligand-binding pocket in 7TM receptors is like a funnel with a partial 'lid...... domains (i.e. especially TM-VI), which performs the large, global toggle switch movements connecting ligand binding with intracellular signaling....

  16. Changes in parathyroid hormone receptor binding affinity during egg laying: implications for calcium homeostasis in chicken.

    Yasuoka, T; Kawashima, M; Takahashi, T; Iwata, A; Oka, N; Tanaka, K

    1996-12-01

    Parathyroid hormone (PTH) receptor bindings were examined in the membrane fraction of the calvaria and the kidney of the hen by the use of [125I]PTH-related protein (PTHrP) binding assays. The binding specificity, reversibility, and saturation of the receptor were demonstrated. The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) were obtained by Scatchard analyses. In both calvaria and kidney, Kd and Bmax values decreased at 3 h before oviposition in egg-laying hens, but not in nonlaying hens. Administration of 17 beta-estradiol or progesterone in vivo caused a decrease in the Kd and Bmax values. Ionized calcium concentrations in the blood plasma showed a decrease at 13 h before oviposition. The results suggest that the PTH receptor binding in the calvaria and the kidney is affected by ovarian steroid hormones and may play a role in maintaining the calcium homeostasis in the egg-laying hen. PMID:8970893

  17. Improved receptor analysis in PET using a priori information from in vitro binding assays

    An accurate determination of non-specific binding is required for the analysis of in vitro and in vivo receptor binding data. For some radioligands the non-specific binding is of the same magnitude as the specific binding. Furthermore, in vitro measurements have shown that the non-specific binding can be different in different brain regions. If this is the case in a PET study for determining Bmax and Kd, a correction for the non-specific binding has to be applied. The aim of the present communication is to present a means for determining corrected Bmax and Kd with Scatchard analysis using in vitro binding studies. The influence of non-specific binding on the free and specifically bound radioligand is expressed with the aid of a correction factor, which can be calculated from measurable quantities. Introduction of the corrected free and specifically bound radioligand should give binding parameters closer to reality than previously obtained results. (author)

  18. Structural analysis of the receptor binding domain of botulinum neurotoxin serotype D

    Zhang, Yanfeng; Buchko, Garry W.; Qin, Lin; Robinson, Howard; Varnum, Susan M.

    2010-10-28

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65 Å resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10 Å relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.

  19. Structural Analysis of the Receptor Binding Domain of Botulinum Neurotoxin Serotype D

    Y Zhang; G Buchko; L Qin; H Robinson; S Varnum

    2011-12-31

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65{angstrom} resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10{angstrom} relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.

  20. Effect of sabcomeline on muscarinic and dopamine receptor binding in intact mouse brain

    Sabcomeline [(R-(Z)-(+)-α-(methoxyiamino)- 1-azabicyclo[2.2.2]octane-3-acetonitrile)] is a potent and functionally selective muscarinic M1 receptor partial agonist. However, little is known of the binding properties of sabcomeline under in vivo conditions. In this study, muscarinic receptor occupancy by sabcomeline in mouse brain regions and heart was estimated using [3H]quinuclidinyl benzilate (QNB) and [3H]N-methylpiperidyl benzilate (NMPB) as radioligands. In the cerebral cortex, hippocampus, and striatum, the estimated IC50 value of sabcomeline for [3H]NMPB binding was almost 0.2 mg/kg. Sabcomeline was not a selective ligand to M1 receptors as compared with biperiden in vivo. In the cerebral cortex, maximum receptor occupancy was observed about 1 hr after intravenous injection of sabcomeline (0.3 mg/kg), and the binding availability of mACh receptors had almost returned to the control level by 3-4 hr. These findings indicated that the binding kinetics of sabcomeline is rather rapid in mouse brain. Examination of dopamine D2 receptor binding revealed that sabcomeline affected the kinetics of both [3H]raclopride and [3H]N-methylspiperone (NMSP) binding in the striatum. It significantly decreased the k3 and k4 of [3H]raclopride binding resulting in an increase in binding potential (BP=k3/k4=Bmax/Kd) in sabcomeline-treated mice, and an approximately 15% decrease in k3 of [3H]NMSP binding was also observed. Although the mechanism is still unclear, sabcomeline altered dopamine D2 receptor affinity or availability by modulations via neural networks. (author)

  1. Kit receptor dimerization is driven by bivalent binding of stem cell factor.

    Lemmon, M A; Pinchasi, D; Zhou, M; Lax, I; Schlessinger, J

    1997-03-01

    Most growth factors and cytokines activate their receptors by inducing dimerization upon binding. We have studied binding of the dimeric cytokine stem cell factor (SCF) to the extracellular domain of its receptor Kit, which is a receptor tyrosine kinase similar to the receptors for platelet-derived growth factor and colony-stimulating factor-1. Calorimetric studies show that one SCF dimer binds simultaneously to two molecules of the Kit extracellular domain. Gel filtration and other methods show that this results in Kit dimerization. It has been proposed that SCF-induced Kit dimerization proceeds via a conformational change that exposes a key receptor dimerization site in the fourth of the five immunoglobulin (Ig)-like domains in Kit. We show that a form of Kit containing just the first three Ig domains (Kit-123) binds to SCF with precisely the same thermodynamic parameters as does Kit-12345. Analytical ultracentrifugation, light scattering, and gel filtration show that Kit-123 dimerizes upon SCF binding in a manner indistinguishable from that seen with Kit-12345. These data argue that the fourth Ig-like domain of Kit is not required for SCF-induced receptor dimerization and provide additional support for a model in which bivalent binding of the SCF dimer provides the driving force for Kit dimerization. PMID:9045650

  2. The different ligand-binding modes of relaxin family peptide receptors RXFP1 and RXFP2.

    Scott, Daniel J; Rosengren, K Johan; Bathgate, Ross A D

    2012-11-01

    Relaxin and insulin-like peptide 3 (INSL3) are peptide hormones with a number of important physiological roles in reproduction, regulation of extracellular matrix turnover, and cardiovascular function. Relaxin and INSL3 mediate their actions through the closely related G-protein coupled receptors, relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2), respectively. These receptors have large extracellular domains (ECD) that contain high-affinity ligand-binding sites within their 10 leucine-rich repeat (LRR)-containing modules. Although relaxin can bind and activate both RXFP1 and RXFP2, INSL3 can only bind and activate RXFP2. To investigate whether this difference is related to the nature of the high-affinity ECD binding site or to differences in secondary binding sites involving the receptor transmembrane (TM) domain, we created a suite of constructs with RXFP1/2 chimeric ECD attached to single TM helices. We show that by changing as little as one LRR, representing four amino acid substitutions, we were able to engineer a high-affinity INSL3-binding site into the ECD of RXFP1. Molecular modeling of the INSL3-RXFP2 interaction based on extensive experimental data highlights the differences in the binding mechanisms of relaxin and INSL3 to the ECD of their cognate receptors. Interestingly, when the engineered RXFP1/2 ECD were introduced into full-length RXFP1 constructs, INSL3 exhibited only low affinity and efficacy on these receptors. These results highlight critical differences both in the ECD binding and in the coordination of the ECD-binding site with the TM domain, and provide new mechanistic insights into the binding and activation events of RXFP1 and RXFP2 by their native hormone ligands. PMID:22973049

  3. Evaluation of the In Vivo and Ex Vivo Binding of Novel BC1 Cannabinoid Receptor Radiotracers

    Miller, A.; Gatley, J.; Gifford, A.

    2002-01-01

    The primary active ingredient of marijuana, 9-tetrahydrocannabinol, exerts its psychoactive effects by binding to cannabinoid CB1 receptors. These receptors are found throughout the brain with high concentrations in the hippocampus and cerebellum. The current study was conducted to evaluate the binding of a newly developed putative cannabinoid antagonist, AM630, and a classical cannabinoid 8-tetrahydrocannabinol as potential PET and/or SPECT imaging agents for brain CB1 receptors. For both of these ligands in vivo and ex vivo studies in mice were conducted. AM630 showed good overall brain uptake (as measure by %IA/g) and a moderately rapid clearance from the brain with a half-clearance time of approximately 30 minutes. However, AM630 did not show selective binding to CB1 cannabinoid receptors. Ex vivo autoradiography supported the lack of selective binding seen in the in vivo study. Similar to AM630, 8-tetrahydrocanibol also failed to show selective binding to CB1 receptor rich brain areas. The 8-tetrahydrocanibol showed moderate overall brain uptake and relatively slow brain clearance as compared to AM630. Further studies were done with AM2233, a cannabinoid ligand with a similar structure as AM630. These studies were done to develop an ex vivo binding assay to quantify the displacement of [131I]AM2233 binding by other ligands in Swiss-Webster and CB1 receptor knockout mice. By developing this assay we hoped to determine the identity of an unknown binding site for AM2233 present in the hippocampus of CB1 knockout mice. Using an approach based on incubation of brain slices prepared from mice given intravenous [131I]AM2233 in either the presence or absence of AM2233 (unlabelled) it was possible to demonstrate a significant AM2233-displacable binding in the Swiss-Webster mice. Future studies will determine if this assay is appropriate for identifying the unknown binding site for AM2233 in the CB1 knockout mice.

  4. Elevated epidermal growth factor (EGF) receptor bindings in dog lung tumors

    The objective of this study is to measure and characterize epidermal growth factor (EGF) receptor binding in dog Pu induced in lung-tumor tissue and to compare then with normal lung tissue in dogs. Epidermal growth factor and its receptors have been shown to play an important role in the proliferation and differentiation of eukaryotic cells. It was therefore hypothesized that inappropriate activation of EGF receptor plays a role in oncogenesis. Microsomal preparations prepared from normal and lung-tumor tissue from beagle dogs were used in a radioreceptor assay, using 125I-EGF as a ligand. Specific EGF receptor bindings were determined in the presence of excess unlabeled EGF. They have examined EGF receptor bindings in 10 lung-tumor samples obtained from seven dogs at necropsy. Epidermal growth factor receptor bindings were significantly higher in eight of the lung-tumor samples compared with both normal lung tissue from control dogs and normal lung tissue from dogs from which tumors were obtained. Their results suggest that the high EGF receptor bindings observed in lung-tumor tissue may play a role in the oncogenesis in dogs

  5. Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin.

    Christoffers, Keith H; Khokhar, Arshia; Chaturvedi, Kirti; Howells, Richard D

    2002-04-15

    We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions. PMID:11853866

  6. Ligand binding was acquired during evolution of nuclear receptors

    Escriva, Hector; Safi, Rachid; Hänni, Catherine; Langlois, Marie-Claire; Saumitou-Laprade, Pierre; Stehelin, Dominique; Capron, André; Pierce, Raymond; Laudet, Vincent

    1997-01-01

    The nuclear receptor (NR) superfamily comprises, in addition to ligand-activated transcription factors, members for which no ligand has been identified to date. We demonstrate that orphan receptors are randomly distributed in the evolutionary tree and that there is no relationship between the position of a given liganded receptor in the tree and the chemical nature of its ligand. NRs are specific to metazoans, as revealed by a screen of NR-related sequences in early- and non-metazoan organism...

  7. Synthesis and Binding Properties of Two New Artificial Anion Receptors

    ZENG Zhen-Ya; HUANG Yan-Yan; HU Ling; WANG Fa-Jun; HE Yong-Bing

    2003-01-01

    @@ The development of anion receptor has attracted increasing interest in supramolecular chemistry, due to poten tial applications in clinical diagnosis, environmental monitoring and biological process. [1] In comparison with thelarge variety of ligands that have been described for cations, [2] the development of selective artificial receptors foranion is still very limited. [3] Two new neutral anion receptors (1 and 2) containing thiourea and amide groups weresynthesized as shown in Scheme 1.

  8. Altered [125I]epidermal growth factor binding and receptor distribution in psoriasis

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [125I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers

  9. Ivermectin binding sites in human and invertebrate Cys-loop receptors

    Lynagh, Timothy Peter; Lynch, Joseph W

    2012-01-01

    modelling now explain how ivermectin binds to these receptors and reveal why it is selective for invertebrate members of the Cys-loop receptor family. Combining this with emerging genomic information, we are now in a position to predict species sensitivity to ivermectin and better understand the molecular...

  10. Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor

    The primary goal of these studies was to localize the steroid-binding and phosphorylated sites of the glucocorticoid receptor. The synthetic steroid, dexamethasone 21-mesylate (DM) forms a covalent thioether bond via the sulfhydryl group of a cysteine residue in the receptor. To determine the covalent site of attachment of this ligand, receptors in WEHI-7 mouse thymoma cells were labeled with [3H]DM and purified with a monoclonal antibody. The receptor was completely digested with trypsin and a single peptide covalently labeled with steroid identified by reversed-phase HPLC. This peptide was analyzed by automated Edman degradation to determine the location of the steroid-labeled residue. A similar analysis was performed on an overlapping peptide produced by Staphylococcus aureus protease digestion. Analysis of tryptic peptides from receptors labeled with both [3H]DM and L-[35S]methionine indicated that this peptide contained methionine. These analyses, coupled with the published amino acid sequence of the receptor, identified Cysteine-644 in the steroid-binding domain of the mouse glucocorticoid receptor as the residue involved in covalent steroid-binding. A synthetic peptide representing amino acids 640-650 of the mouse receptor was prepared and analyzed to confirm the identification. These biochemical studies represent a direct demonstration of an amino acid important in receptor function. It has been proposed that the receptor functions through a phosphorylation-dephosphorylation cycle to explain the dependence of hormone binding capacity upon cellular ATP. The glucocorticoid receptor has been shown to be a phosphoprotein. As an initial step to identifying a role of phosphorylation in receptor action, phosphorylated sites within the functional domains of the protein were identified

  11. The Different Ligand-Binding Modes of Relaxin Family Peptide Receptors RXFP1 and RXFP2

    Scott, Daniel J.; Rosengren, K. Johan; Bathgate, Ross A. D.

    2012-01-01

    Relaxin and insulin-like peptide 3 (INSL3) are peptide hormones with a number of important physiological roles in reproduction, regulation of extracellular matrix turnover, and cardiovascular function. Relaxin and INSL3 mediate their actions through the closely related G-protein coupled receptors, relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2), respectively. These receptors have large extracellular domains (ECD) that contain high-affinity ligand-binding sites within their 10 leuci...

  12. Structural Studies of GABAA Receptor Binding Sites: Which Experimental Structure Tells us What?

    Puthenkalam, Roshan; Hieckel, Marcel; Simeone, Xenia; Suwattanasophon, Chonticha; Feldbauer, Roman V; Ecker, Gerhard F; Ernst, Margot

    2016-01-01

    Atomic resolution structures of cys-loop receptors, including one of a γ-aminobutyric acid type A receptor (GABAA receptor) subtype, allow amazing insights into the structural features and conformational changes that these pentameric ligand-gated ion channels (pLGICs) display. Here we present a comprehensive analysis of more than 30 cys-loop receptor structures of homologous proteins that revealed several allosteric binding sites not previously described in GABAA receptors. These novel binding sites were examined in GABAA receptor homology models and assessed as putative candidate sites for allosteric ligands. Four so far undescribed putative ligand binding sites were proposed for follow up studies based on their presence in the GABAA receptor homology models. A comprehensive analysis of conserved structural features in GABAA and glycine receptors (GlyRs), the glutamate gated ion channel, the bacterial homologs Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus GLIC, and the serotonin type 3 (5-HT3) receptor was performed. The conserved features were integrated into a master alignment that led to improved homology models. The large fragment of the intracellular domain that is present in the structure of the 5-HT3 receptor was utilized to generate GABAA receptor models with a corresponding intracellular domain fragment. Results of mutational and photoaffinity ligand studies in GABAA receptors were analyzed in the light of the model structures. This led to an assignment of candidate ligands to two proposed novel pockets, candidate binding sites for furosemide and neurosteroids in the trans-membrane domain were identified. The homology models can serve as hypotheses generators, and some previously controversial structural interpretations of biochemical data can be resolved in the light of the presented multi-template approach to comparative modeling. Crystal and cryo-EM microscopic structures of the closest homologs that were solved in different conformational

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

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

    2016-01-01

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

  14. Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

    Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125I-insulin specific binding was not affected. The decrease in 125I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/105 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125I-epidermal growth factor binding. (au)

  15. Computational approaches to modeling receptor flexibility upon ligand binding: Application to interfacially activated enzymes

    Wade, R.C.; Sobolev, V.; Ortiz, A.R. .;

    1998-01-01

    Receptors generally undergo conformational change upon ligand binding. We describe how fairly simple techniques may be used in docking and design studies to account for some of the changes in the conformations of proteins on ligand binding. Simulations of protein-ligand interactions that give a m...... a more complete description of the dynamics important for ligand binding are then discussed. These methods are illustrated for phospholipase A(2) and lipase, enzymes that both undergo interfacial activation....

  16. Reduced 5-HT2A receptor binding in patients with mild cognitive impairment

    Hasselbalch, S G; Madsen, K; Svarer, C;

    2008-01-01

    cerebral 5-HT(2A) receptor binding in patients with mild cognitive impairment (MCI) and related 5-HT(2A) receptor binding to clinical symptoms. Sixteen patients with MCI of the amnestic type (mean age 73, mean MMSE 26.1) and 17 age and sex matched control subjects were studied with MRI and [(18)F......Previous studies of patients with Alzheimer's disease (AD) have described reduced brain serotonin 2A (5-HT(2A)) receptor density. It is unclear whether this abnormality sets in early in the course of the disease and whether it is related to early cognitive and neuropsychiatric symptoms. We assessed...

  17. Point mutations of human interleukin-1 with decreased receptor binding affinity

    Interleukin-1 (IL-1) is a monocyte-derived polypeptide hormone that interacts with a plasma membrane receptor. We have used oligonucleotide-directed mutagenesis to construct mutant human IL-1 proteins. Three different point mutants in a unique histidine residue (position 30) exhibited varying degrees of reduced IL-1 receptor binding affinity, whereas point mutants at five other residues behaved normally. Structural analysis of these mutant proteins by nuclear magnetic resonance spectroscopy detected no (or only minor) conformational changes relative to wild-type IL-1. These data suggest that the unique histidine residue influences the architecture of the receptor binding site on human IL-1. (Auth.)

  18. Aberrant Expression of Functional BAFF-System Receptors by Malignant B-Cell Precursors Impacts Leukemia Cell Survival

    Maia, Sara; Pelletier, Marc; Ding, Jixin; Hsu, Yen-Ming; Rao, Sambasiva P.; Cardoso, Angelo A.; Sallan, Stephen Earl; Nadler, Lee Marshall

    2011-01-01

    Despite exhibiting oncogenic events, patient's leukemia cells are responsive and dependent on signals from their malignant bone marrow (BM) microenvironment, which modulate their survival, cell cycle progression, trafficking and resistance to chemotherapy. Identification of the signaling pathways mediating this leukemia/microenvironment interplay is critical for the development of novel molecular targeted therapies. We observed that primary leukemia B-cell precursors aberrantly express recept...

  19. The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1

    Sethi, Ashish; Bruell, Shoni; Patil, Nitin; Hossain, Mohammed Akhter; Scott, Daniel J.; Petrie, Emma J.; Bathgate, Ross A. D.; Gooley, Paul R.

    2016-01-01

    H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the L...

  20. Binding and uptake of H-ferritin are mediated by human transferrin receptor-1.

    Li, Li; Fang, Celia J; Ryan, James C; Niemi, Eréne C; Lebrón, José A; Björkman, Pamela J; Arase, Hisashi; Torti, Frank M; Torti, Suzy V; Nakamura, Mary C; Seaman, William E

    2010-02-23

    Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules. PMID:20133674

  1. Identification of a Binding Site for Unsaturated Fatty Acids in the Orphan Nuclear Receptor Nurr1.

    de Vera, Ian Mitchelle S; Giri, Pankaj K; Munoz-Tello, Paola; Brust, Richard; Fuhrmann, Jakob; Matta-Camacho, Edna; Shang, Jinsai; Campbell, Sean; Wilson, Henry D; Granados, Juan; Gardner, William J; Creamer, Trevor P; Solt, Laura A; Kojetin, Douglas J

    2016-07-15

    Nurr1/NR4A2 is an orphan nuclear receptor, and currently there are no known natural ligands that bind Nurr1. A recent metabolomics study identified unsaturated fatty acids, including arachidonic acid and docosahexaenoic acid (DHA), that interact with the ligand-binding domain (LBD) of a related orphan receptor, Nur77/NR4A1. However, the binding location and whether these ligands bind other NR4A receptors were not defined. Here, we show that unsaturated fatty acids also interact with the Nurr1 LBD, and solution NMR spectroscopy reveals the binding epitope of DHA at its putative ligand-binding pocket. Biochemical assays reveal that DHA-bound Nurr1 interacts with high affinity with a peptide derived from PIASγ, a protein that interacts with Nurr1 in cellular extracts, and DHA also affects cellular Nurr1 transactivation. This work is the first structural report of a natural ligand binding to a canonical NR4A ligand-binding pocket and indicates a natural ligand can bind and affect Nurr1 function. PMID:27128111

  2. Characterization of a second ligand binding site of the insulin receptor

    Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the α subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K d of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site

  3. GABA binding to an insect GABA receptor: a molecular dynamics and mutagenesis study.

    Ashby, Jamie A; McGonigle, Ian V; Price, Kerry L; Cohen, Netta; Comitani, Federico; Dougherty, Dennis A; Molteni, Carla; Lummis, Sarah C R

    2012-11-21

    RDL receptors are GABA-activated inhibitory Cys-loop receptors found throughout the insect CNS. They are a key target for insecticides. Here, we characterize the GABA binding site in RDL receptors using computational and electrophysiological techniques. A homology model of the extracellular domain of RDL was generated and GABA docked into the binding site. Molecular dynamics simulations predicted critical GABA binding interactions with aromatic residues F206, Y254, and Y109 and hydrophilic residues E204, S176, R111, R166, S176, and T251. These residues were mutated, expressed in Xenopus oocytes, and their functions assessed using electrophysiology. The data support the binding mechanism provided by the simulations, which predict that GABA forms many interactions with binding site residues, the most significant of which are cation-π interactions with F206 and Y254, H-bonds with E204, S205, R111, S176, T251, and ionic interactions with R111 and E204. These findings clarify the roles of a range of residues in binding GABA in the RDL receptor, and also show that molecular dynamics simulations are a useful tool to identify specific interactions in Cys-loop receptors. PMID:23200041

  4. Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

    Song, Ge; Shao, Xiao-Xia; Wu, Qing-Ping; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand–receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand–receptor interaction studies. PMID:27414797

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

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

    2015-09-01

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

  6. Ivermectin binding sites in human and invertebrate Cys-loop receptors.

    Lynagh, Timothy; Lynch, Joseph W

    2012-08-01

    Ivermectin is a gold standard antiparasitic drug that has been used successfully to treat billions of humans, livestock and pets. Until recently, the binding site on its Cys-loop receptor target had been a mystery. Recent protein crystal structures, site-directed mutagenesis data and molecular modelling now explain how ivermectin binds to these receptors and reveal why it is selective for invertebrate members of the Cys-loop receptor family. Combining this with emerging genomic information, we are now in a position to predict species sensitivity to ivermectin and better understand the molecular basis of ivermectin resistance. An understanding of the molecular structure of the ivermectin binding site, which is formed at the interface of two adjacent subunits in the transmembrane domain of the receptor, should also aid the development of new lead compounds both as anthelmintics and as therapies for a wide variety of human neurological disorders. PMID:22677714

  7. Effect of estradiol-17β on calcitonin receptor bindings in the hen neurohypophysis.

    Nakayama, H; Takahashi, T; Nakagawa-Mizuyachi, K; Kawashima, M

    2011-01-01

    The present study was performed to elucidate whether estradiol-17β (E₂) would affect calcitonin (CT) receptor binding in the hen neurohypophysis. The equilibrium dissociation constant (K(d)) and the maximum binding capacity (B(max)) of the CT receptor in the plasma membrane fraction of the hen neurohypophysis were examined by Scatchard analysis of specific binding of (125)I-labeled chicken CT. A single i.m. injection of E₂ into nonlaying hens caused a decrease in K(d) and B(max) values of the CT receptor. The K(d) and B(max) values of the CT receptor were smaller in laying hens than in nonlaying hens. The present study suggests that E₂ may increase the action of CT on the neurohypophysis in hens. PMID:21177459

  8. Sedative-Hypnotic and Receptor Binding Studies of Fermented Marine Organisms.

    Joung, Hye-Young; Kang, Young Mi; Lee, Bae-Jin; Chung, Sun Yong; Kim, Kyung-Soo; Shim, Insop

    2015-09-01

    This study was performed to investigate the sedative-hypnotic activity of γ-aminobutyric acid (GABA)-enriched fermented marine organisms (FMO), including sea tangle (FST) and oyster (FO) by Lactobacillus brevis BJ20 (L. brevis BJ20). FST and FO were tested for their binding activity of the GABAA-benzodiazepine and 5-HT2C receptors, which are well-known molecular targets for sleep aids. We also measured the sleep latency and sleep duration during pentobarbital-induced sleep in mice after oral administration of FST and FO. In GABAA and 5-HT2C receptor binding assays, FST displayed an effective concentration-dependent binding affinity to GABAA receptor, similar to the binding affinity to 5-HT2C receptor. FO exhibited higher affinity to 5-HT2C receptor, compared with the GABAA receptor. The oral administration of FST and FO produced a dose-dependent decrease in sleep latency and increase in sleep duration in pentobarbital-induced hypnosis. The data demonstrate that FST and FO possess sedative-hypnotic activity possibly by modulating GABAA and 5-HT2C receptors. We propose that FST and FO might be effective agents for treatment of insomnia. PMID:26336589

  9. Thermodynamic Characterization of New Positive Allosteric Modulators Binding to the Glutamate Receptor A2 Ligand-Binding Domain

    Nørholm, Ann-Beth; Francotte, Pierre; Goffin, Eric; Botez, Iuliana; Danober, Laurence; Lestage, Pierre; Pirotte, Bernard; Kastrup, Jette Sandholm Jensen; Olsen, Lars; Oostenbrink, Chris

    2014-01-01

    5a (5-F) and 5b (6-F) are entropy driven. For 5d (8-F), both quantities were equal in size. Thermodynamic integration (TI) and one-step perturbation (OSP) were used to calculate the relative binding affinity of the modulators. The OSP calculations had a higher predictive power than those from TI......Positive allosteric modulation of the ionotropic glutamate receptor GluA2 presents a potential treatment of cognitive disorders, for example, Alzheimer's disease. In the present study, we describe the synthesis, pharmacology, and thermodynamic studies of a series of monofluoro-substituted 3......,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides. Measurements of ligand binding by isothermal titration calorimetry (ITC) showed similar binding affinities for the modulator series at the GluA2 LBD but differences in the thermodynamic driving forces. Binding of 5c (7-F) and 6 (no-F) is enthalpy driven, and...

  10. Multiple opioid receptor binding in dissociated intact guinea pig brain cells

    Tam, S.W.; James, D.W.

    1986-03-05

    Dissociated intact guinea pig brain cells were prepared by the method of Rogers and El-Fakahany. Over 95% of these cells are viable as demonstrated by their exclusion of the dye trypan blue. Opioid receptor binding assays were performed in a modified Kreb-Ringers physiological buffer. The following radiolabeled ligands and conditions were used to selectively labeled multiple opioid receptors: mu binding, 1 nM (/sup 3/H)naloxone + 20 nM DADLE + 300 nM U50,488H; kappa binding, 4 nM (-)-(/sup 3/H)-EKC + 100 nM DAGO + 500 nM DADLE; delta binding, 2 nM (/sup 3/H)-DADLE + 100 nM DAGO + 300 nM U50,488H; sigma binding, 4 nM (+)-(/sup 3/H)SKF 10,047. The intact brain cells in physiological buffer demonstrated specific binding for mu, kappa, delta, and sigma receptors. The relative binding potency of naloxone for each of the receptor types is arbitrarily set at 1.

  11. Multiple opioid receptor binding in dissociated intact guinea pig brain cells

    Dissociated intact guinea pig brain cells were prepared by the method of Rogers and El-Fakahany. Over 95% of these cells are viable as demonstrated by their exclusion of the dye trypan blue. Opioid receptor binding assays were performed in a modified Kreb-Ringers physiological buffer. The following radiolabeled ligands and conditions were used to selectively labeled multiple opioid receptors: mu binding, 1 nM [3H]naloxone + 20 nM DADLE + 300 nM U50,488H; kappa binding, 4 nM (-)-[3H]-EKC + 100 nM DAGO + 500 nM DADLE; delta binding, 2 nM [3H]-DADLE + 100 nM DAGO + 300 nM U50,488H; sigma binding, 4 nM (+)-[3H]SKF 10,047. The intact brain cells in physiological buffer demonstrated specific binding for mu, kappa, delta, and sigma receptors. The relative binding potency of naloxone for each of the receptor types is arbitrarily set at 1

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

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

    2016-01-01

    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-RTAH/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. PMID:27353000

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

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

    2013-01-01

    . falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins was...... unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and...... group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology and the...

  14. Alterations in the structure and DNA binding of Ah receptor in the presence of polyamines

    Polyamines, putrescine, spermidine, and spermine are organic cations present in all cells. They have multiple regulatory roles in cell growth and differentiation. 2,3,7,8-Tetrachlorodigenzo-p-dioxin (TCDD), and related polycyclic aromatic hydrocarbons exert their adverse effects by binding to an intracellular protein (Ah receptor). Interaction of Ah receptor with specific DNA sequences triggers gene regulatory effects of TCDD. The authors examined the effect of polyamines on the structure and DNA binding of Ah receptor isolated from a human squamous carcinoma cell line, A431. [3H]TCDD-labeled Hf receptor was sedimented in the 9S region of sucrose gradients in hypotenic buffer. Polyamines caused a concentration dependent condensation and precipitation of Ah receptor. In the presence of 1 mM spermine the receptor was completely precipitated which could be recovered from the bottom of the sucrose gradients. This precipitation did not occur with RNase-treated Ah receptor. Incubation of RNase-treated Ah receptor with 1 mM spermidine increased its DNA binding 10-fold compared to controls having equivalent ionic strength. These results suggest that endogenous polyamines may influence the structural organization and gene regulatory effects of TCDD

  15. Syntheses of 7-Substituted α-Cyperone Derivatives for Selective Sigma-1 Receptor over Cannabinoid-1 Receptor Binding Affinities

    We have successfully synthesized seven α-cyperone derivatives and found that the presence of a hydrogen bond donor/acceptor groups at the C7 position of α-cyperone significantly affects specificity and potency of CB1 receptor binding affinity over sigma-1 receptor binding affinity. In particular, the presence of the amino moiety at the C7 position of α-cyperone is beneficial for binding to sigmia-1 receptor. The molecular mechanism of compound 8 involved in the high binding affinity to sigma-1 receptor is under investigation. We first synthesized α-cyperone 1 by following the previously reported synthetic routes.15-19 In brief, azeotropic imination of (+)-dihydrocarvone and (R)-(+)-1-phenylethylamine followed by alkylation with a slight excess of ethyl vinyl ketone (EVK) in THF at 40 .deg. C produced the Micheal adduct. The resulting adduct was hydrolyzed and then treated with sodium methoxide at room temperature to give an easily separable mixture of α-cyperone 1 and its side product. Flash chromatography resulted in pure α-cyperone 1 in a 30% yield from (+)-dihydrocarvone

  16. Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

    Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai; Tao, Yong-guang; Tolbert, W. David; Simons, Jr., S. Stoney; Xu, H. Eric (NIH)

    2010-03-08

    A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

  17. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

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

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

    2003-01-01

    of receptor function and in turn for the design and development of novel therapeutic compound. Here we show how ligand-receptor interaction can be investigated in situ with high sensitivity on sensor surfaces by total internal reflection fluorescence (TIRF) measurements. A generally applicable method...... streptavidin. TIRF measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without...

  19. Differential utilization of binding loop flexibility in T cell receptor ligand selection and cross-reactivity.

    Ayres, Cory M; Scott, Daniel R; Corcelli, Steven A; Baker, Brian M

    2016-01-01

    Complementarity determining region (CDR) loop flexibility has been suggested to play an important role in the selection and binding of ligands by T cell receptors (TCRs) of the cellular immune system. However, questions remain regarding the role of loop motion in TCR binding, and crystallographic structures have raised questions about the extent to which generalizations can be made. Here we studied the flexibility of two structurally well characterized αβ TCRs, A6 and DMF5. We found that the two receptors utilize loop motion very differently in ligand binding and cross-reactivity. While the loops of A6 move rapidly in an uncorrelated fashion, those of DMF5 are substantially less mobile. Accordingly, the mechanisms of binding and cross-reactivity are very different between the two TCRs: whereas A6 relies on conformational selection to select and bind different ligands, DMF5 uses a more rigid, permissive architecture with greater reliance on slower motions or induced-fit. In addition to binding site flexibility, we also explored whether ligand-binding resulted in common dynamical changes in A6 and DMF5 that could contribute to TCR triggering. Although binding-linked motional changes propagated throughout both receptors, no common features were observed, suggesting that changes in nanosecond-level TCR structural dynamics do not contribute to T cell signaling. PMID:27118724

  20. HTLV-1 and -2 envelope SU subdomains and critical determinants in receptor binding

    Valle Carine

    2004-12-01

    Full Text Available Abstract Background Human T-cell leukemia virus (HTLV -1 and -2 are deltaretroviruses that infect a wide range of cells. Glut1, the major vertebrate glucose transporter, has been shown to be the HTLV Env receptor. While it is well established that the extracellular surface component (SU of the HTLV envelope glycoprotein (Env harbors all of the determinants of interaction with the receptor, identification of SU subdomains that are necessary and sufficient for interaction with the receptor, as well as critical amino acids therein, remain to be precisely defined. Although highly divergent in the rest of their genomes, HTLV and murine leukemia virus (MLV Env appear to be related and based on homologous motifs between the HTLV and MLV SU, we derived chimeric HTLV/MLV Env and soluble HTLV-1 and -2 truncated amino terminal SU subdomains. Results Using these SU constructs, we found that the 183 and 178 amino terminal residues of the HTLV-1 and -2 Env, respectively, were sufficient to efficiently bind target cells of different species. Binding resulted from bona fide interaction with the HTLV receptor as isolated SU subdomains specifically interfered with HTLV Env-mediated binding, cell fusion, and cell-free as well as cell-to-cell infection. Therefore, the HTLV receptor-binding domain (RBD lies in the amino terminus of the SU, immediately upstream of a central immunodominant proline rich region (Env residues 180 to 205, that we show to be dispensible for receptor-binding and interference. Moreover, we identified a highly conserved tyrosine residue at position 114 of HTLV-1 Env, Tyr114, as critical for receptor-binding and subsequent interference to cell-to-cell fusion and infection. Finally, we observed that residues in the vicinity of Tyr114 have lesser impact on receptor binding and had various efficiency in interference to post-binding events. Conclusions The first 160 residues of the HTLV-1 and -2 mature cleaved SU fold as autonomous domains that

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

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-05-01

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

  2. Opioid binding properties of the purified kappa receptor from human placenta

    A glycoprotein with a molecular weight of 63,000 has been purified, in an active form, from human placental villus tissue membranes. The binding properties of this glycoprotein to opioid alkaloids and peptides indicates that it is the kappa opiate receptor of human placenta. The receptor binds the tritiated ligands etorphine, bremazocine, ethylketocyclazocine and naloxone specifically and reversibly with Kd values of 3.3, 4.4, 5.1 and 7.0nM, respectively. The binding of 3H-Bremazocine to the purified receptor is inhibited by the following compounds with the corresponding Ki values EKC, 1.3 x 10-8M; Dynorphin 1-8, 3.03 x 10-7; U50,488H, 4.48 x 10-9; U69-593,2.28 x 10-8, morphine, 4.05 x 10-6 DADLE, 6.47 x 10-6 and naloxone, 2.64 x 10-8. The purified receptor binds 8 nmole of 3H-Etorphine and 1.7 nmole 3H-BZC per mg protein. The theoretical binding capacity of a protein of this molecular weight is 15.8. Although the iodinated purified receptor appears by autoradiography as one band on SDS-PAGE, yet homogeneity of the preparation is not claimed

  3. Opioid binding properties of the purified kappa receptor from human placenta

    Ahmed, M.S.; Zhou, D.; Cavinato, A.G.; Maulik, D.

    1989-01-01

    A glycoprotein with a molecular weight of 63,000 has been purified, in an active form, from human placental villus tissue membranes. The binding properties of this glycoprotein to opioid alkaloids and peptides indicates that it is the kappa opiate receptor of human placenta. The receptor binds the tritiated ligands etorphine, bremazocine, ethylketocyclazocine and naloxone specifically and reversibly with Kd values of 3.3, 4.4, 5.1 and 7.0nM, respectively. The binding of /sup 3/H-Bremazocine to the purified receptor is inhibited by the following compounds with the corresponding Ki values EKC, 1.3 x 10/sup -8/M; Dynorphin 1-8, 3.03 x 10/sup -7/; U50,488H, 4.48 x 10/sup -9/; U69-593,2.28 x 10/sup -8/, morphine, 4.05 x 10/sup -6/ DADLE, 6.47 x 10/sup -6/ and naloxone, 2.64 x 10/sup -8/. The purified receptor binds 8 nmole of /sup 3/H-Etorphine and 1.7 nmole /sup 3/H-BZC per mg protein. The theoretical binding capacity of a protein of this molecular weight is 15.8. Although the iodinated purified receptor appears by autoradiography as one band on SDS-PAGE, yet homogeneity of the preparation is not claimed.

  4. Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

    Owczarek, C M; Layton, M. J.; Metcalf, D; Lock, P; Willson, T A; Gough, N M; Nicola, N A

    1993-01-01

    Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mappe...

  5. The role of antigenically different virus neuraminidases as structures implicated in receptor-binding processes.

    Coimbra, M V; Luiz, M O; Cabral, M C; Couceiro, J N

    1995-06-01

    Influenza A viruses exhibit segmented nucleic acid coding for eight different proteins, two of them as glycoproteins exposed on their lipoprotein envelopes, hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin exhibits receptor-binding activity while neuraminidase develops sialidase cleavage activity which acts on cell receptors. Influenza A strains responsible for human, avian, equine and porcine respiratory infections all over the world present antigenically different hemagglutinin (H1 to H14) and neuraminidase (N1 to N9) structures on their surface. The objective of the present investigation was to study the role of N2, N8, and N9, antigenically diverse neuraminidase structures of human (N2) and animal (N8 and N9) influenza viruses, in the receptor-binding process. Receptor-binding activity of N2 and N8 was analyzed by crossed tests using H3N2 and H3N8 antisera and the hemagglutination inhibition test as a model. Hemagglutinating activity of antigenically different N2 and N8 structures was demonstrable and was inhibited by homologous antisera (N2-H3N2, N8-H3N8) but not by heterologous antisera (N2-H3N8,N8-H3N2). This previously demonstrated N9 hemagglutinating activity was analyzed for receptor-binding specificity using hemagglutination tests and NeuAc alpha2,3Gal and NeuAc alpha2,6Gal derivatized erythrocytes. This highly purified N9 strain was obtained from a virus strain isolated from terns by Dr. Peter Colman (CSIRO Division of Biomolecular Engineering, Parkville, Victoria, Australia). It exhibited receptor-binding specificity for NeuAc alpha2,3Gal sequences, a property similar to that observed in hemagglutinins from avian strains. These results indicate the importance of antigenically different neuraminidase structures as alternative agents for developing receptor-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8547843

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

    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, 3H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a 3H-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 3H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A4, 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

  7. MANAGING TIGHT BINDING RECEPTORS FOR NEW SPEARATIONS TECHNOLOGIES

    DARYLE H BUSCH RICHARD S GIVENS

    2004-12-10

    Much of the earth's pollution involves compounds of the metallic elements, including actinides, strontium, cesium, technetium, and RCRA metals. Metal ions bind to molecules called ligands, which are the molecular tools that can manipulate the metal ions under most conditions. This DOE-EMSP sponsored program strives (1) to provide the foundations for using the most powerful ligands in transformational separations technologies and (2) to produce seminal examples of their applications to separations appropriate to the DOE EM mission. These ultra tight-binding ligands can capture metal ions in the most competitive of circumstances (from mineralized sites, lesser ligands, and even extremely dilute solutions), but they react so slowly that they are useless in traditional separations methodologies. Two attacks on this problem are underway. The first accommodates to the challenging molecular lethargy by developing a seminal slow separations methodology termed the soil poultice. The second designs ligands that are only tight-binding while wrapped around the targeted metal ion, but can be put in place by switch-binding and removed by switch-release. We envision a kind of molecular switching process to accelerate the union between metal ion and tight-binding ligand. Molecular switching processes are suggested for overcoming the slow natural equilibration rate with which ultra tight-binding ligands combine with metal ions. Ligands that bind relatively weakly combine with metal ions rapidly, so the trick is to convert a ligand from a weak, rapidly binding species to a powerful, slow releasing ligand--during the binding of the ligand to the metal ion. Such switch-binding ligands must react with themselves, and the reaction must take place under the influence of the metal ion. For example, our generation 1 ligands showed that a well-designed linear ligand with ends that readily combine, forms a cyclic molecule when it wraps around a metal ion. Our generation 2 ligands are

  8. Aging-induced changes in brain regional serotonin receptor binding: Effect of Carnosine.

    Banerjee, S; Poddar, M K

    2016-04-01

    Monoamine neurotransmitter, serotonin (5-HT) has its own specific receptors in both pre- and post-synapse. In the present study the role of carnosine on aging-induced changes of [(3)H]-5-HT receptor binding in different brain regions in a rat model was studied. The results showed that during aging (18 and 24 months) the [(3)H]-5-HT receptor binding was reduced in hippocampus, hypothalamus and pons-medulla with a decrease in their both Bmax and KD but in cerebral cortex the [(3)H]-5-HT binding was increased with the increase of its only Bmax. The aging-induced changes in [(3)H]-5-HT receptor binding with carnosine (2.0 μg/kg/day, intrathecally, for 21 consecutive days) attenuated in (a) 24-month-aged rats irrespective of the brain regions with the attenuation of its Bmax except hypothalamus where both Bmax and KD were significantly attenuated, (b) hippocampus and hypothalamus of 18-month-aged rats with the attenuation of its Bmax, and restored toward the [(3)H]-5-HT receptor binding that observed in 4-month-young rats. The decrease in pons-medullary [(3)H]-5-HT binding including its Bmax of 18-month-aged rats was promoted with carnosine without any significant change in its cerebral cortex. The [(3)H]-5-HT receptor binding with the same dosages of carnosine in 4-month-young rats (a) increased in the cerebral cortex and hippocampus with the increase in their only Bmax whereas (b) decreased in hypothalamus and pons-medulla with a decrease in their both Bmax and KD. These results suggest that carnosine treatment may (a) play a preventive role in aging-induced brain region-specific changes in serotonergic activity (b) not be worthy in 4-month-young rats in relation to the brain regional serotonergic activity. PMID:26808776

  9. Elevated epidermal growth factor receptor binding in plutonium-induced lung tumors from dogs

    The objective of this study is to examine and characterize epidermal growth factor receptor (EGF-R) binding in inhaled plutonium-induced canine lung-tumor tissue and to compare it with that in normal canine lung tissue. Crude membrane preparations from normal and lung-tumor tissue from beagle dogs were examined in a radioreceptor assay, using 125I-labeled epidermal growth factor (EGF) as a ligand. Specific EGF receptor binding was determined in the presence of excess unlabeled EGF. We have examined EGF receptor binding in eight lung-tumor samples obtained from six dogs. Epidermal growth factor receptor binding was significantly greater in lung-tumor samples (31.38%) compared with that in normal lung tissue (3.76%). Scatchard plot analysis from the displacement assay revealed that there was no statistical difference in the binding affinity but significantly higher concentration of EGF-R sites in the lung-tumor tissue (619 fmol/mg) than in normal lung tissue (53 fmol/mg). The increase in EGF-R number in plutonium-induced dog lung tumors does not seem to correlate with increase in the initial lung burden exposure to plutonium. Our results demonstrate that there is a significant increase in EGF-R binding in inhaled plutonium-induced dog lung tumors

  10. Demonstration of beta1-adrenergic receptors in human placenta by (-)I125 Iodocyanopindolol binding

    The highly specific β-adrenergic radioligand (-)125I Iodocyanopindolol (ICYP) was used to characterize the β-adrenergic receptor subtype present in human placenta. Binding of ICYP to membranes from human placenta was saturable with time and ligand concentration, of high affinity, and demonstrated appropriate stereoselectivity and agonist rank order of potency for binding to a β-adrenergic receptor. From saturation binding curves, the KD and Bmax values for ICYP binding were 233±51 pM and 690±139 fmol/mg of proteins, respectively.Analysis of inhibition of ICYP binding by β1- and β2-selective adrenergic antagonists via Hofstee analysis resulted in linear plots, indicating the existence of a homogeneous population of β-adrenergic receptors. From the resulting KI-values for the β1-selective drugs practolol (4.0±0.9 μM) and metoprolol (0.19±0.07 μM) and for the β2-selective drug ICI 118,551 (0.30)±0.06 μM) it is concluded that the β-adrenergic receptor in human placenta is of the β1-subtype. This is further supported by the fact that (-)-noradrenaline and (-)-adrenaline were equipotent in inhibiting ICYP binding

  11. Evaluation of a novel virtual screening strategy using receptor decoy binding sites.

    Patel, Hershna; Kukol, Andreas

    2016-01-01

    Virtual screening is used in biomedical research to predict the binding affinity of a large set of small organic molecules to protein receptor targets. This report shows the development and evaluation of a novel yet straightforward attempt to improve this ranking in receptor-based molecular docking using a receptor-decoy strategy. This strategy includes defining a decoy binding site on the receptor and adjusting the ranking of the true binding-site virtual screen based on the decoy-site screen. The results show that by docking against a receptor-decoy site with Autodock Vina, improved Receiver Operator Characteristic Enrichment (ROCE) was achieved for 5 out of fifteen receptor targets investigated, when up to 15 % of a decoy site rank list was considered. No improved enrichment was seen for 7 targets, while for 3 targets the ROCE was reduced. The extent to which this strategy can effectively improve ligand prediction is dependent on the target receptor investigated. PMID:27553084

  12. A mollusk retinoic acid receptor (RAR) ortholog sheds light on the evolution of ligand binding.

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Lima, Daniela; Pierzchalski, Keely; Jones, Jace W; Kane, Maureen; Nishikawa, Jun-Ichi; Hiromori, Youhei; Nakanishi, Tsuyoshi; Santos, Miguel M; Castro, L Filipe C; Bourguet, William; Schubert, Michael; Laudet, Vincent

    2014-11-01

    Nuclear receptors are transcription factors that regulate networks of target genes in response to small molecules. There is a strong bias in our knowledge of these receptors because they were mainly characterized in classical model organisms, mostly vertebrates. Therefore, the evolutionary origins of specific ligand-receptor couples still remain elusive. Here we present the identification and characterization of a retinoic acid receptor (RAR) from the mollusk Nucella lapillus (NlRAR). We show that this receptor specifically binds to DNA response elements organized in direct repeats as a heterodimer with retinoid X receptor. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid or any other retinoid we tested. Furthermore, NlRAR is unable to activate the transcription of reporter genes in response to stimulation by retinoids and to recruit coactivators in the presence of these compounds. Three-dimensional modeling of the ligand-binding domain of NlRAR reveals an overall structure that is similar to vertebrate RARs. However, in the ligand-binding pocket (LBP) of the mollusk receptor, the alteration of several residues interacting with the ligand has apparently led to an overall decrease in the strength of the interaction with the ligand. Accordingly, mutations of NlRAR at key positions within the LBP generate receptors that are responsive to retinoids. Altogether our data suggest that, in mollusks, RAR has lost its affinity for all-trans retinoic acid, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of nuclear receptors validated by millions of years of evolution that were impossible to reveal in model organisms. PMID:25116705

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

    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

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

    Gatfield, John; Mueller Grandjean, Celia; Bur, Daniel; Bolli, Martin H; Nayler, Oliver

    2014-01-01

    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 insurmountable

  15. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1.

    Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues. PMID:27583834

  16. Ascorbic acid enables reversible dopamine receptor /sup 3/H-agonist binding

    Leff, S.; Sibley, D.R.; Hamblin, M.; Creese, I.

    1981-11-16

    The effects of ascorbic acid on dopaminergic /sup 3/H-agonist receptor binding were studied in membrane homogenates of bovine anterior pituitary and caudate, and rat striatum. In all tissues virtually no stereospecific binding (defined using 1uM (+)butaclamol) of the /sup 3/H-agonists N-propylnorapomorphine (NPA), apomorphine, or dopamine could be demonstrated in the absence of ascorbic acid. Although levels of total /sup 3/H-agonist binding were three to five times greater in the absence than in the presence of 0.1% ascorbic acid, the increased binding was entirely non-stereospecific. Greater amounts of dopamine-inhibitable /sup 3/H-NPA binding could be demonstrated in the absence of 0.1% ascorbic acid, but this measure of ''specific binding'' was demonstrated not to represent dopamine receptor binding since several other catecholamines and catechol were equipotent with dopamine and more potent than the dopamine agonist (+/-)amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) in inhibiting this binding. High levels of dopamine-displaceable /sup 3/H-agonist binding were detected in fresh and boiled homogenates of cerebellum, an area of brain which receives no dopaminergic innervation, further demonstrating the non-specific nature of /sup 3/H-agonist binding in the absence of ascorbic acid. These studies emphasize that under typical assay conditions ascorbic acid is required in order to demonstrate reversible and specific /sup 3/H-agonist binding to dopamine receptors.

  17. Interleukin 1α and interleukin 1β bind to the same receptor on T cells

    Pure, E. coli-derived recombinant murine interleukin 1α (IL 1α) was labeled with 125I and used for receptor binding studies. The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner. Scatchard plot analysis for binding studies carried out at 40C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10-10 M and the presence of approximately 1200 binding sites per cell. Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas interferon-αA, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect. The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface. The authors have also examined the ability of purified recombinant human IL 1α and IL 1β to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells. They report here that both human IL 1 proteins are able to recognize the same binding site on mouse IL 1. In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells

  18. In vivo (/sup 3/H)flunitrazepam binding: imaging of receptor regulation

    Ciliax, B.J.; Penney, J.B. Jr.; Young, A.B.

    1986-08-01

    The use of (/sup 3/H)flunitrazepam as a ligand to measure alterations in benzodiazepine receptors in vivo in rats was investigated. Animals were injected with (/sup 3/H)flunitrazepam i.v., arterial samples of (/sup 3/H)flunitrazepam were obtained and, later, the animals were sacrificed to assay brain binding. (/sup 3/H)flunitrazepam enters the brain rapidly and binds to benzodiazepine receptors. About two-thirds of this binding is blocked by predosing the animals with 5 mg/kg of clonazepam. The amount of remaining (nonspecific) binding correlates very well (r = 0.88) with the amount of radioactivity found in plasma at the time of death. A series of rats were lesioned unilaterally with kainic acid in the caudate-putamen several months before the infusion of (/sup 3/H)flunitrazepam. In vivo autoradiography in lesioned rats showed that benzodiazepine binding in globus pallidus and substantia nigra on the side of the lesion was increased significantly as compared to the intact side. The observed changes in benzodiazepine binding were similar to those observed previously in lesioned rats using in vitro techniques. Thus, benzodiazepine receptor regulation can be imaged quantitatively using in vivo binding techniques.

  19. In vivo [3H]flunitrazepam binding: imaging of receptor regulation

    The use of [3H]flunitrazepam as a ligand to measure alterations in benzodiazepine receptors in vivo in rats was investigated. Animals were injected with [3H]flunitrazepam i.v., arterial samples of [3H]flunitrazepam were obtained and, later, the animals were sacrificed to assay brain binding. [3H]flunitrazepam enters the brain rapidly and binds to benzodiazepine receptors. About two-thirds of this binding is blocked by predosing the animals with 5 mg/kg of clonazepam. The amount of remaining (nonspecific) binding correlates very well (r = 0.88) with the amount of radioactivity found in plasma at the time of death. A series of rats were lesioned unilaterally with kainic acid in the caudate-putamen several months before the infusion of [3H]flunitrazepam. In vivo autoradiography in lesioned rats showed that benzodiazepine binding in globus pallidus and substantia nigra on the side of the lesion was increased significantly as compared to the intact side. The observed changes in benzodiazepine binding were similar to those observed previously in lesioned rats using in vitro techniques. Thus, benzodiazepine receptor regulation can be imaged quantitatively using in vivo binding techniques

  20. Modulatory effects of unsaturated fatty acids on the binding of glucocorticoids to rat liver glucocorticoid receptors.

    Vallette, G; Vanet, A; Sumida, C; Nunez, E A

    1991-09-01

    Binding of the synthetic glucocorticoid dexamethasone to the rat liver cytosol glucocorticoid receptor was inhibited by physiological concentrations of nonesterified fatty acids as a function of increasing dose, degree of unsaturation, and chain length of the fatty acid. Polyunsaturated fatty acids were the most potent inhibitors. Scatchard analysis and Line-weaver-Burk plots of the binding data revealed that both the association constants and number of binding sites decreased and that polyunsaturated fatty acids inhibition was of a mixed non-competitive type. The dissociation rate constant of [3H]dexamethasone from glucocorticoid receptors was increased by up to 10 times in the presence of docosahexaenoic acid, whereas a competitive inhibitor like the glucocorticoid antagonist RU 38486 had no effect. Moreover, sucrose density gradient analysis showed that docosahexaenoic acid inhibited the binding of [3H] dexamethasone to both the 8.8S and 4S forms. The results strongly suggest that unsaturated fatty acids are interacting at a site on the receptor different from the hormone binding site and the heat shock protein and that by binding to a second site unsaturated fatty acids greatly change the conformation of the hormone binding site to reduce its affinity for the hormone, either partially or completely depending on the concentration and the class of the fatty acid. PMID:1874175

  1. Miniaturizing VEGF: Peptides mimicking the discontinuous VEGF receptor-binding site modulate the angiogenic response

    De Rosa, Lucia; Finetti, Federica; Diana, Donatella; di Stasi, Rossella; Auriemma, Sara; Romanelli, Alessandra; Fattorusso, Roberto; Ziche, Marina; Morbidelli, Lucia; D’Andrea, Luca Domenico

    2016-08-01

    The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.

  2. Maps of receptor binding parameters in the human brain - a kinetic analysis of pet measurements

    A kinetic method is described for the estimation of neuroreceptor density as well as the rate constants for association and dissociation of rapidly equilibrating radioligands. The method is exemplified by positron emission tomographic measurements of the human brain using 11C-raclopride, a D2 dopamine receptor antagonist, and 11C-Ro 15-1788, a benzodiazepine receptor antagonist. Using a linear non iterative algorithm, regional binding characteristics were calculated and displayed poixel by pixel in brain maps. Data from repeated experiments on the same subject with different amounts of the unlabeled ligand were utilized. The binding characteristics were determined according to a two step procedure in which the time course of the free radioligand concentration was estimated from a reference region considered to be free of specific receptor binding sites. Alternative methods to determine the concentration of free radioligand are discussed. (orig.)

  3. Biological activity of cloned mammary tumor virus DNA fragments that bind purified glucocorticoid receptor protein in vitro

    To test whether high-affinity receptor:DNA interactions can be correlated with receptor effects on promoter function in vivo, we have mapped in greater detail the receptor-binding regions on murine mammary tumor virus DNA, using both nitrocellulose-filter binding and electron microscopy. Recombinant plasmids bearing these receptor-binding domains have been transfected into cultured cells, and the expression of the plasmid sequences has been monitored for hormonal regulation. The results are considered in terms of a speculative proposal that the glucocorticoid receptor may effect changes in promoter activity via specific alteration of chromatin and/or DNA structure. 37 references, 6 figures, 2 tables

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

    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.

  5. Relative binding affinity of diethylstilbestrol to uterine nuclear estrogen receptor: effect of serum and serum albumin

    The relative binding affinity (RBA) of tritium labelled diethylstilbestrol (DES) was determined in nuclear fractions of rat uterus. DES displayed a two- to threefold greater affinity than estradiol for nuclear E receptor. The RBA of DES to nuclear E receptor was lowered significantly in the presence of rat serum (43 +/- 1) or human serum (52 +/- 7). Dilution of human serum resulted in a progressive increase in the RBA of DES which approached that observed in the absence of serum. Addition of purified human serum albumin mimicked the decrease in RBA of DES that was observed with serum. The IC50 of estradiol was not changed in the presence of either rat serum or albumin. These data show that DES possesses a greater affinity for nuclear E receptor than estradiol and that serum albumin can modulate DES binding to uterine E receptor

  6. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

    Nie,Y.; Hobbs, J.; Vigues, S.; Olson, W.; Conn, G.; Munger, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.

  7. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, [3H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [3H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites

  8. Temporal cortex dopamine D2/3 receptor binding in major depression.

    Lehto, Soili M; Kuikka, Jyrki; Tolmunen, Tommi; Hintikka, Jukka; Viinamäki, Heimo; Vanninen, Ritva; Haatainen, Kaisa; Koivumaa-Honkanen, Heli; Honkalampi, Kirsi; Tiihonen, Jari

    2008-06-01

    The aim of this study was to assess the dopamine function of the temporal cortex in major depressive disorder using [(123)I]epidepride to image D(2/3) receptor binding sites. Ten major depressives and 10 healthy controls were selected from a general population sample for single-photon emission computed tomography imaging. Among the major depressives there was a strong bilateral correlation between the scores on the 21-item Hamilton Depression Rating Scale and D(2/3) receptor binding. Dopaminergic abnormalities may be present in the temporal cortices of major depressives. PMID:18588596

  9. Synthesis and receptor binding affinity of new selective GluR5 ligands

    Bunch, L; Johansen, T H; Bräuner-Osborne, Hans;

    2001-01-01

    Two hybrid analogues of the kainic acid receptor agonists, 2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA) and (2S,4R)-4-methylglutamic acid ((2S,4R)-4-Me-Glu), were designed, synthesized, and characterized in radioligand binding assays using cloned ionotropic and metabotropi.......0 and 2.0 microM. respectively. Their affinities in the [3H]AMPA binding assay on native cortical receptors were shown to correlate with their GluR2 affinity rather than their GluR5 affinity. No affinity for GluR6 was detected (IC50 > 100 microM)....

  10. Ligand-induced Coupling versus Receptor Pre-association: Cellular automaton simulations of FGF-2 binding

    Gopalakrishnan, Manoj; Forsten-Williams, Kimberly; Tauber, Uwe C.

    2003-01-01

    The binding of basic fibroblast growth factor (FGF-2) to its cell surface receptor (CSR) and subsequent signal transduction is known to be enhanced by Heparan Sulfate Proteoglycans (HSPGs). HSPGs bind FGF-2 with low affinity and likely impact CSR-mediated signaling via stabilization of FGF-2-CSR complexes via association with both the ligand and the receptor. What is unknown is whether HSPG associates with CSR in the absence of FGF-2. In this paper, we determine conditions by which pre-associ...

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

    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

  12. Genome-Wide Binding and Transcriptome Analysis of Human Farnesoid X Receptor in Primary Human Hepatocytes

    Zhan, Le; Liu, Hui-Xin; Fang, Yaping; Kong, Bo; He, Yuqi; Zhong, Xiao-bo; Fang, Jianwen; Wan, Yu-Jui Yvonne; Guo, Grace L.

    2014-01-01

    Background & Aims Farnesoid X receptor (FXR, NR1H4) is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing...

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

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

    2013-01-01

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

  14. Cytotoxic purine nucleoside analogues bind to A1, A2A and A3 adenosine receptors

    Jensen, Kyle; Johnson, L’Aurelle A.; Jacobson, Pamala A.; Kachler, Sonja; Kirstein, Mark N.; Lamba, Jatinder; Klotz, Karl-Norbert

    2012-01-01

    Fludarabine, clofarabine and cladribine are anti-cancer agents which are analogues of the purine nucleoside adenosine. These agents have been associated with cardiac and neurological toxicities. Because these agents are analogues of adenosine, they may act through adenosine receptors to elicit their toxic effects. The objective of this study was to evaluate the ability of cytotoxic nucleoside analogues to bind and activate adenosine receptor subtypes (A1, A2A, A2B, and A3). Radioligand bindin...

  15. Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

    Molinari, A M; Abbondanza, C; Armetta, I.; Medici, N; Minucci, S; Moncharmont, B; Nigro, V; Puca, G A

    1991-01-01

    The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl...

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

    Tony Velkov

    2013-01-01

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

  17. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

    Pfreundschuh, Moritz; Alsteens, David; Wieneke, Ralph; Zhang, Cheng; Coughlin, Shaun R.; Tampé, Robert; Kobilka, Brian K.; Müller, Daniel J.

    2015-11-01

    A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

  18. Tension-compression asymmetry in the binding affinity of membrane-anchored receptors and ligands

    Xu, Guang-Kui; Liu, Zishun; Feng, Xi-Qiao; Gao, Huajian

    2016-03-01

    Cell adhesion plays a crucial role in many biological processes of cells, e.g., immune responses, tissue morphogenesis, and stem cell differentiation. An essential problem in the molecular mechanism of cell adhesion is to characterize the binding affinity of membrane-anchored receptors and ligands under different physiological conditions. In this paper, a theoretical model is presented to study the binding affinity between a large number of anchored receptors and ligands under both tensile and compressive stresses, and corroborated by demonstrating excellent agreement with Monte Carlo simulations. It is shown that the binding affinity becomes lower as the magnitude of the applied stress increases, and drops to zero at a critical tensile or compressive stress. Interestingly, the critical compressive stress is found to be substantially smaller than the critical tensile stress for relatively long and flexible receptor-ligand complexes. This counterintuitive finding is explained by using the Euler instability theory of slender columns under compression. The tension-compression asymmetry in the binding affinity of anchored receptors and ligands depends subtly on the competition between the breaking and instability of their complexes. This study helps in understanding the role of mechanical forces in cell adhesion mediated by specific binding molecules.

  19. Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor.

    Maillet, Emeline L; Cui, Meng; Jiang, Peihua; Mezei, Mihaly; Hecht, Elizabeth; Quijada, Jeniffer; Margolskee, Robert F; Osman, Roman; Max, Marianna

    2015-10-01

    The sweet taste receptor, a heterodimeric G protein-coupled receptor comprised of T1R2 and T1R3, binds sugars, small molecule sweeteners, and sweet proteins to multiple binding sites. The dipeptide sweetener, aspartame binds in the Venus Flytrap Module (VFTM) of T1R2. We developed homology models of the open and closed forms of human T1R2 and human T1R3 VFTMs and their dimers and then docked aspartame into the closed form of T1R2's VFTM. To test and refine the predictions of our model, we mutated various T1R2 VFTM residues, assayed activity of the mutants and identified 11 critical residues (S40, Y103, D142, S144, S165, S168, Y215, D278, E302, D307, and R383) in and proximal to the binding pocket of the sweet taste receptor that are important for ligand recognition and activity of aspartame. Furthermore, we propose that binding is dependent on 2 water molecules situated in the ligand pocket that bridge 2 carbonyl groups of aspartame to residues D142 and L279. These results shed light on the activation mechanism and how signal transmission arising from the extracellular domain of the T1R2 monomer of the sweet receptor leads to the perception of sweet taste. PMID:26377607

  20. Taste substance binding elicits conformational change of taste receptor T1r heterodimer extracellular domains.

    Nango, Eriko; Akiyama, Shuji; Maki-Yonekura, Saori; Ashikawa, Yuji; Kusakabe, Yuko; Krayukhina, Elena; Maruno, Takahiro; Uchiyama, Susumu; Nuemket, Nipawan; Yonekura, Koji; Shimizu, Madoka; Atsumi, Nanako; Yasui, Norihisa; Hikima, Takaaki; Yamamoto, Masaki; Kobayashi, Yuji; Yamashita, Atsuko

    2016-01-01

    Sweet and umami tastes are perceived by T1r taste receptors in oral cavity. T1rs are class C G-protein coupled receptors (GPCRs), and the extracellular ligand binding domains (LBDs) of T1r1/T1r3 and T1r2/T1r3 heterodimers are responsible for binding of chemical substances eliciting umami or sweet taste. However, molecular analyses of T1r have been hampered due to the difficulties in recombinant expression and protein purification, and thus little is known about mechanisms for taste perception. Here we show the first molecular view of reception of a taste substance by a taste receptor, where the binding of the taste substance elicits a different conformational state of T1r2/T1r3 LBD heterodimer. Electron microscopy has showed a characteristic dimeric structure. Förster resonance energy transfer and X-ray solution scattering have revealed the transition of the dimerization manner of the ligand binding domains, from a widely spread to compactly organized state upon taste substance binding, which may correspond to distinct receptor functional states. PMID:27160511

  1. In vivo receptor binding of opioid drugs at the mu site

    The in vivo receptor binding of a series of opioid drugs was investigated in intact rats after s.c. administration of [3H]etorphine tracer, which selectively binds to mu sites in vivo. Receptor binding was determined by a membrane filtration assay immediately after sacrifice of the animals and brain homogenization. Coadministration of unlabeled opioid drugs together with tracer led to a dose-dependent decrease of in vivo tracer binding. Estimates of the doses required to occupy 50% of the mu sites in vivo established the following potency rank order: diprenorphine, naloxone, buprenorphine, etorphine, levallorphan, cyclazocine, sufentanil, nalorphine, ethylketocyclazocine, ketocyclazocine, pentazocine, morphine. In vivo-in vitro differences among the relative receptor binding potencies were only partially accounted for by differences in their access to the brain and the regulatory effects of Na+ and GTP, which are expected to reduce agonist affinities in vivo. The relationship among mu receptor occupancy in vivo and pharmacological effects of the opioid drugs is described

  2. Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain

    Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of 125I-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for 125I-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. 125I-M110 and 125I-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL was comparable to that of 125I-oPRL by unlabeled oPRL, while 125I-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I-M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82

  3. MDM2 binds and inhibits vitamin D receptor

    Heyne, Kristina; Heil, Tessa-Carina; Bette, Birgit; Reichrath, Jörg; Roemer, Klaus

    2015-01-01

    The E3 ubiquitin ligase and transcriptional repressor MDM2 is a potent inhibitor of the p53 family of transcription factors and tumor suppressors. Herein, we report that vitamin D receptor (VDR), another transcriptional regulator and probably, tumor suppressor, is also bound and inhibited by MDM2. This interaction was not affected by vitamin D ligand. VDR was ubiquitylated in the cell and its steady-state level was controlled by the proteasome. Strikingly, overproduced MDM2 reduced the level ...

  4. Thermodynamics of calmodulin binding to cardiac and skeletal muscle ryanodine receptor ion channels

    Meissner, Gerhard; Pasek, Daniel A.; Yamaguchi, Naohiro; Ramachandran, Srinivas; Dokholyan, Nikolay V.; Tripathy, Ashutosh

    2009-01-01

    The skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptor calcium release channels contain a single, conserved calmodulin (CaM) binding domain, yet are differentially regulated by CaM. Here, we report that high-affinity [35S]CaM binding to RyR1 is driven by favorable enthalpic and entropic contributions at Ca2+ concentrations from

  5. The glucocorticoid receptor hormone binding domain mediates transcriptional activation in vitro in the absence of ligand.

    Schmitt, J.; Stunnenberg, H G

    1993-01-01

    We show that recombinant rat glucocorticoid receptor (vvGR) expressed using vaccinia virus is indistinguishable from authentic GR with respect to DNA and hormone binding. In the absence of hormone, vvGR is mainly found in the cytoplasm in a complex with heat shock protein 90. Upon incubation with ligand, vvGR is released from this complex and translocated to the nucleus. Thus, the ligand binding domain displays the known biochemical properties. However, in vitro, transcription from a syntheti...

  6. Receptor-mediated binding of C1 q on pulmonary endothelial cells

    Normal undamaged pulmonary endothelial cells (EC) do not express receptors for C3b or the Fc portion of IgG, but both receptors become unmasked after viral infection or exposure to white cell lysates. Here, highly purified human C1q was labeled with 125I, and the globular subunits were separated from the collagenous portion by collagenase digestion and chromatography. Bovine pulmonary artery EC were cultured without exposure to proteolytic enzymes. Binding assays were carried out at 00C, pH 7.4, μ = 0.15. Results showed that 125I-C1q binds to EC, the binding is dose-dependent, and the receptor is saturable. Saturation was approached at a C1q concentration of ca 0.1 ug. By Scatchard analysis, maximum binding was 0.219 pmoles in a volume of 250 μl for 7 x 105 cells, and the average number of binding sites per cell was 1.88 x 105. Isolated 125I-C1q heads do not bind, and when native 125I-C1q was bound to EC radioactivity was eliminated after collagenase treatment for 4 h at 370C. Thus, C1q binds to EC via the collagenous portion. That Fc receptors (globular heads) are exposed was shown by rosette formation with EA and EC bound C1q. Using similar conditions, native C1(C1w x 2C1r x 2C1s) did not bind to EC. These results suggest a mechanism for localizing immune complexes on undamaged pulmonary vessels which may be important for initiation of the inflammatory response

  7. Androgen regulation of epidermal growth factor receptor binding activity during fetal rabbit lung development.

    Klein, J M; Nielsen, H C

    1993-01-01

    Fetal lung development progresses in a sex-specific manner with male fetuses exhibiting delayed maturation. Androgens, both exogenous and endogenous, inhibit while epidermal growth factor (EGF) enhances fetal lung development. We hypothesized that one mechanism responsible for the delay in male fetal lung development is an androgen-induced delay in EGF receptor binding activity. We measured EGF binding in sex-specific fetal rabbit lung plasma membranes isolated from control fetuses (days 21, ...

  8. Receptor-like function of heparin in the binding and uptake of neutral lipids.

    Bosner, M S; Gulick, T; Riley, D J; Spilburg, C. A.; Lange, L G

    1988-01-01

    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 ...

  9. Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites

    Kaae, Birgitte Høiriis; Harpsøe, Kasper; Kastrup, Jette Sandholm Jensen;

    2007-01-01

    have dramatically increased potencies, more than three orders of magnitude higher than the corresponding monomers. Dimer (R,R)-2a was cocrystallized with the GluR2-S1S2J construct, and an X-ray crystallographic analysis showed (R,R)-2a to bridge two identical binding pockets on two neighboring GluR2...... subunits. Thus, this is biostructural evidence of a homomeric dimer bridging two identical receptor-binding sites....

  10. In vivo binding of [11C]nemonapride to sigma receptors in the cortex and cerebellum

    Radiolabeled nemonapride (NEM, YM-09151-2) is widely used as a representative dopamine D2-like receptor ligand in pharmacological and neurological studies, and 11C-labeled analog ([11C]NEM) has been developed for positron emission tomography (PET) studies. The aim of this study was to evaluate whether [11C]NEM binds in vivo to sigma receptors. [11C]NEM and one of six dopamine D2-like receptor ligands or seven sigma receptor ligands were co-injected into mice, and the regional brain uptake of [11C]NEM was measured by a tissue dissection method. The striatal uptake of [11C]NEM was reduced by D2-like receptor ligands, NEM, haloperidol, (+)-butaclamol, raclopride, and sulpiride, but not by a D4 receptor ligand clozapine. In the cortex and cerebellum the uptake was also reduced by D2-like receptor ligands with affinity for sigma receptors, but not by raclopride. Although none of seven sigma receptor ligands, SA6298, N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine hydrochloride (NE-100), (+)-pentazocine, R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane hydrochloride ([-]-PPAP), (-)-pentazocine, R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ([+]-3-PPP), and (+)-N-allylnormetazocine hydrochloride ([+]-SKF 10047), blocked the striatal uptake, five of them with relatively higher affinity significantly reduced the [11C]NEM uptake by the cortex, and four of them reduced that by the cerebellum. We concluded that [11C]NEM binds in vivo not only to dopamine D2-like receptors in the striatum but also to sigma receptors in other regions such as cortex and cerebellum

  11. An amphioxus orthologue of the estrogen receptor that does not bind estradiol: Insights into estrogen receptor evolution

    Laudet Vincent

    2008-07-01

    Full Text Available Abstract Background The origin of nuclear receptors (NRs and the question whether the ancestral NR was a liganded or an unliganded transcription factor has been recently debated. To obtain insight into the evolution of the ligand binding ability of estrogen receptors (ER, we comparatively characterized the ER from the protochordate amphioxus (Branchiostoma floridae, and the ER from lamprey (Petromyzon marinus, a basal vertebrate. Results Extensive phylogenetic studies as well as signature analysis allowed us to confirm that the amphioxus ER (amphiER and the lamprey ER (lampER belong to the ER group. LampER behaves as a "classical" vertebrate ER, as it binds to specific DNA Estrogen Responsive Elements (EREs, and is activated by estradiol (E2, the classical ER natural ligand. In contrast, we found that although amphiER binds EREs, it is unable to bind E2 and to activate transcription in response to E2. Among the 7 natural and synthetic ER ligands tested as well as a large repertoire of 14 cholesterol derivatives, only Bisphenol A (an endocrine disruptor with estrogenic activity bound to amphiER, suggesting that a ligand binding pocket exists within the receptor. Parsimony analysis considering all available ER sequences suggest that the ancestral ER was not able to bind E2 and that this ability evolved specifically in the vertebrate lineage. This result does not support a previous analysis based on ancestral sequence reconstruction that proposed the ancestral steroid receptor to bind estradiol. We show that biased taxonomic sampling can alter the calculation of ancestral sequence and that the previous result might stem from a high proportion of vertebrate ERs in the dataset used to compute the ancestral sequence. Conclusion Taken together, our results highlight the importance of comparative experimental approaches vs ancestral reconstructions for the evolutionary study of endocrine systems: comparative analysis of extant ERs suggests that the

  12. Effect of receptor binding domain mutations on receptor binding and transmissibility of avian influenza H5N1 viruses

    Maines, Taronna R; Chen, Li-Mei; Van Hoeven, Neal; Tumpey, Terrence M; Blixt, Ola; Belser, Jessica A; Gustin, Kortney M; Pearce, Melissa B; Pappas, Claudia; Stevens, James; Cox, Nancy J; Paulson, James C; Raman, Rahul; Sasisekharan, Ram; Katz, Jacqueline M; Donis, Ruben O

    2011-01-01

    Although H5N1 influenza viruses have been responsible for hundreds of human infections, these avian influenza viruses have not fully adapted to the human host. The lack of sustained transmission in humans may be due, in part, to their avian-like receptor preference. Here, we have introduced...

  13. Time course of the estradiol-dependent induction of oxytocin receptor binding in the ventromedial hypothalamic nucleus of the rat

    Johnson, A.E.; Ball, G.F.; Coirini, H.; Harbaugh, C.R.; McEwen, B.S.; Insel, T.R. (National Institute of Mental Health, Poolesville, MD (USA))

    1989-09-01

    Oxytocin (OT) transmission is involved in the steroid-dependent display of sexual receptivity in rats. One of the biochemical processes stimulated by the ovarian steroid 17 beta-estradiol (E2) that is relevant to reproduction is the induction of OT receptor binding in the ventromedial hypothalamic nucleus (VMN). The purpose of these experiments was to determine if E2-induced changes in OT receptor binding in the VMN occur within a time frame relevant to cyclic changes in ovarian steroid secretion. OT receptor binding was measured in the VMN of ovariectomized rats implanted for 0-96 h with E2-containing Silastic capsules. The rate of decay of OT receptor binding was measured in another group of animals 6-48 h after capsule removal. Receptors were labeled with the specific OT receptor antagonist ({sup 125}I)d(CH2)5(Tyr(Me)2,Thr4,Tyr-NH2(9))OVT, and binding was measured with quantitative autoradiographic methods. In addition, plasma E2 levels and uterine weights were assessed in animals from each treatment condition. Significant increases in E2-dependent OT receptor binding and uterine weight occurred within 24 h of steroid treatment. After E2 withdrawal, OT receptor binding and uterine weight decreased significantly within 24 h. These results are consistent with the hypothesis that steroid modulation of OT receptor binding is necessary for the induction of sexual receptivity.

  14. Bio-inspired Dynamic Gradients Regulated by Supramolecular Bindings in Receptor-Embedded Hydrogel Matrices.

    Luan, Xinglong; Zhang, Yihe; Wu, Jing; Jonkheijm, Pascal; Li, Guangtao; Jiang, Lei; Huskens, Jurriaan; An, Qi

    2016-08-01

    The kinetics of supramolecular bindings are fundamentally important for molecular motions and spatial-temporal distributions in biological systems, but have rarely been employed in preparing artificial materials. This report proposes a bio-inspired concept to regulate dynamic gradients through the coupled supramolecular binding and diffusion process in receptor-embedded hydrogel matrices. A new type of hydrogel that uses cyclodextrin (CD) as both the gelling moiety and the receptors is prepared as the diffusion matrices. The diffusible guest, 4-aminoazobenzene, quickly and reversibly binds to matrices-bound CD during diffusion and generates steeper gradients than regular diffusion. Weakened bindings induced through UV irradiation extend the gradients. Combined with numerical simulation, these results indicate that the coupled binding-diffusion could be viewed as slowed diffusion, regulated jointly by the binding constant and the equilibrium receptor concentrations, and gradients within a bio-relevant extent of 4 mm are preserved up to 90 h. This report should inspire design strategies of biomedical or cell-culturing materials. PMID:27547643

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

    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.

  16. Putative hAPN receptor binding sites in SARS_CoV spike protein

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

    2003-01-01

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

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

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

  18. Monoclonal antibodies to the human insulin receptor block insulin binding and inhibit insulin action.

    Roth, R A; Cassell, D J; Wong, K. Y.; Maddux, B A; Goldfine, I D

    1982-01-01

    Antibodies to the insulin receptor were prepared in BALB/c mice by immunization with IM-9 human lymphocytes, a cell type that has a large number of plasma membrane insulin receptors. The spleens of these mice were then removed, and their lymphocytes were fused to a mouse myeloma cell line, FO cells. After screening over 1,200 resulting hybrids, one stable hybrid was obtained that produced IgG1 antibodies directed towards the insulin receptor. This antibody blocked 125I-labeled insulin binding...

  19. Cloning, ligand-binding, and temporal expression of ecdysteroid receptors in the diamondback moth, Plutella xylostella

    Tang Baozhen

    2012-10-01

    Full Text Available Abstract Background The diamondback moth, Plutella xylostella (L. (Lepidoptera: Plutellidae, is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors. Result In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro-translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM. In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous. Conclusions Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists to their targets (ecdysone receptors leads to an adaptive response (resistance, is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella.

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

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

    2003-01-01

    Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity, and soluble heparan sulfate blocked infection of cells by wild-type pseudovirus. Interestingly, some but not all MMTV-like elements found in primary and cultured human breast cancer cell lines, termed h-MTVs, had sequence alterations in the putative RBS. Single substitution of one of the amino acids found in an h-MTV RBS variant in the RBD of MMTV, Phe40 to Ser, did not alter species tropism but abolished both virus binding to cells and infectivity. Neutralizing anti-SU monoclonal antibodies also recognized a glutathione S-transferase fusion protein that contained the five-amino-acid RBS region from MMTV. The critical Phe40 residue is located on a surface of the MMTV RBD model that is distant from and may be structurally more rigid than the region of F-MLV RBD that contains its critical binding site residues. This suggests that, in contrast to other murine retroviruses, binding to its receptor may result in few or no changes in MMTV envelope protein conformation. PMID:12970432

  1. The ligand specificities of the insulin receptor and the insulin-like growth factor I receptor reside in different regions of a common binding site

    Kjeldsen, T.; Andersen, A.S.; Wiberg, F.C.; Rasmussen, J.S.; Schaeffer, L.; Balschmidt, P.; Moller, K.B.; Moller, N.P.H. (Novo Nordisk, Bagsvaerd (Denmark))

    1991-05-15

    To identify the region(s) of the insulin receptor and the insulin-like growth factor I (IGF-I) receptor responsible for ligand specificity (high-affinity binding), expression vectors encoding soluble chimeric insulin/IGF-I receptors were prepared. The chimeric receptors were expressed in mammalian cells and partially purified. Binding studies revealed that a construct comprising an IGF-I receptor in which the 68 N-terminal amino acids of the insulin receptor {alpha}-subunit had replaced the equivalent IGF-I receptor segment displayed a markedly increased affinity for insulin. In contrast, the corresponding IGF-I receptor sequence is not critical for high-affinity IGF-I binding. It is shown that part of the cysteine-rich domain determines IGF-I specificity. The authors have previously shown that exchanging exons 1, 2, and 3 of the insulin receptor with the corresponding IGF-I receptor sequence results in loss of high affinity for insulin and gain of high affinity for IGF-I. Consequently, it is suggested that the ligand specificities of the two receptors (i.e., the sequences that discriminate between insulin and IGF-I) reside in different regions of a binding site with common features present in both receptors.

  2. Xanomeline binding to and activation of muscarinic receptors

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

    Pianoro: Medimond, 2009 - (Fisher, A.; Hanin, I.), s. 145-148 ISBN 978-88-7587-528-2. [International Conference Alzheimer ´s diseases/Parkinson´s diseases /9./. Praha (CZ), 11.05.2009-15.05.2009] R&D Projects: GA AV ČR(CZ) IAA500110703; GA ČR GA305/09/0681; GA MŠk(CZ) LC554 Grant ostatní: EC(XE) LiPiDiDiet 211696 Institutional research plan: CEZ:AV0Z50110509 Keywords : xanomeline * muscarinic receptors * signal transduction Subject RIV: FH - Neurology

  3. Anti-idiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor

    Anti-idiotypic antibodies were raised in rabbits by immunization with a monoclonal anti-haloperidol antibody. Some of these anti-idiotypic antibodies bind in a concentration dependent manner to bovine striatal membranes. Following affinity purification, these antibodies inhibit haloperidol binding to striatal membranes and deplete [3H]-spiperone binding sites from a solubilized preparation of striatal membranes. It is thus concluded that these anti-idiotypic antibodies are an internal image of haloperidol and as such can interact with D2-dopamine receptors

  4. Receptor binding site-deleted foot-and-mouth disease (FMD) virus protects cattle from FMD.

    McKenna, T S; Lubroth, J; Rieder, E; Baxt, B; Mason, P W

    1995-01-01

    Binding of foot-and-mouth disease virus (FMDV) to cells requires an arginine-glycine-aspartic acid (RGD) sequence in the capsid protein VP1. We have genetically engineered an FMDV in which these three amino acids have been deleted, producing a virus particle which is unable to bind to cells. Cattle vaccinated with these receptor binding site-deleted virions were protected from disease when challenged with a virulent virus, demonstrating that these RGD-deleted viruses could serve as the basis ...

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

    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....

  6. Chronic morphine treatment up-regulates mu opioid receptor binding in cells lacking Filamin A

    Onoprishvili, Irma; Simon, Eric J.

    2007-01-01

    We investigated the effects of morphine and other agonists on the human mu opioid receptor (MOP) expressed in M2 melanoma cells, lacking the actin cytoskeleton protein filamin A and in A7, a sub clone of the M2 melanoma cells, stably transfected with filamin A cDNA. The results of binding experiments showed, that after chronic morphine treatment (24 hr) of A7 cells, MOP binding sites were down-regulated to 63% of control, whereas, unexpectedly, in M2 cells, MOP binding was up-regulated to 188...

  7. Evidence That the Thyroid-stimulating Hormone (TSH) Receptor Transmembrane Domain Influences Kinetics of TSH Binding to the Receptor Ectodomain*

    Chen, Chun-Rong; McLachlan, Sandra M.; Rapoport, Basil

    2010-01-01

    Thyroid-stimulating hormone (TSH)-induced reduction in ligand binding affinity (negative cooperativity) requires TSH receptor (TSHR) homodimerization, the latter involving primarily the transmembrane domain (TMD) but with the extracellular domain (ECD) also contributing to this association. To test the role of the TMD in negative cooperativity, we studied the TSHR ECD tethered to the cell surface by a glycosylphosphatidylinositol (GPI) anchor that multimerizes despite the absence of the TMD. ...

  8. Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies.

    Bokori-Brown, Monika; Kokkinidou, Maria C; Savva, Christos G; Fernandes da Costa, Sérgio; Naylor, Claire E; Cole, Ambrose R; Moss, David S; Basak, Ajit K; Titball, Richard W

    2013-05-01

    Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity. PMID:23504825

  9. Changes in angiotensin II receptor bindings in the hen neurohypophysis before and after oviposition.

    Takahashi, T; Nozaki, Y; Nakagawa-Mizuyachi, K; Nakayama, H; Kawashima, M

    2011-11-01

    The present study was performed to elucidate whether the angiotensin II (ANG II) receptor exists in the plasma membrane fraction of the neurohypophysis in hens, to estimate the time of action of ANG II on the neurohypophysis before and after oviposition, and to examine relationships between the action of ANG II on the neurohypophysis and those of estrogen and prostaglandin F(2α) (PGF(2α)) in relation to arginine vasotocin (AVT) release. The specific binding had a binding specificity to chicken ANG II (cANG II), reversibility, and saturation in the [(125)I]cANG II binding assay. Scatchard analysis revealed that the binding sites are of a single class. The equilibrium dissociation constant (K(d)) obtained by kinetic analysis and Scatchard analysis suggested a high affinity, and the maximum binding capacity (B(max)) obtained by Scatchard analysis suggested a limited capacity. These results suggest that an ANG II receptor exists in the neurohypophysis of hens. The K(d) and the B(max) value was significantly smaller in laying hens than in nonlaying hens, which suggests that bindings of the cANG II receptor change, depending on the difference in laying condition. Values of the K(d) and the B(max) decreased approximately 15 min before oviposition in laying hens, and decreased 1 h after an intramuscular injection of estradiol-17β and 5 min after an intravenous injection of cANG II in nonlaying hens. The amount of specific binding of PGF(2α) receptor in the neurohypophysis also decreased and AVT concentration in blood increased after the cANG II injection. It seems likely that the action of cANG II in the neurohypophysis increases due to the effect of estrogen approximately 15 min before oviposition, and the cANG II action stimulates AVT release through the increase in the PGF(2α) action in this tissue. PMID:22010242

  10. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

    Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TRα1, TRβ1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TRα1, TRβ1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

  11. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

    Istrate, Monica A., E-mail: monicai@scripps.edu [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany); Nussler, Andreas K., E-mail: nuessler@uchir.me.tum.de [Department of Traumatology, Technical University Munich, Ismaningerstr. 22, 81675 Munich (Germany); Eichelbaum, Michel, E-mail: michel.eichelbaum@ikp-stuttgart.de [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany); Burk, Oliver, E-mail: oliver.burk@ikp-stuttgart.de [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany)

    2010-03-19

    Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

  12. Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

    Pinkney, M; Hoggett, J G

    1988-03-15

    Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase. PMID:2839152

  13. Glycan receptor binding of the influenza A virus H7N9 hemagglutinin.

    Tharakaraman, Kannan; Jayaraman, Akila; Raman, Rahul; Viswanathan, Karthik; Stebbins, Nathan W; Johnson, David; Shriver, Zachary; Sasisekharan, V; Sasisekharan, Ram

    2013-06-20

    The advent of H7N9 in early 2013 is of concern for a number of reasons, including its capability to infect humans, the lack of clarity in the etiology of infection, and because the human population does not have pre-existing immunity to the H7 subtype. Earlier sequence analyses of H7N9 hemagglutinin (HA) point to amino acid changes that predicted human receptor binding and impinge on the antigenic characteristics of the HA. Here, we report that the H7N9 HA shows limited binding to human receptors; however, should a single amino acid mutation occur, this would result in structural changes within the receptor binding site that allow for extensive binding to human receptors present in the upper respiratory tract. Furthermore, a subset of the H7N9 HA sequences demarcating coevolving amino acids appears to be in the antigenic regions of H7, which, in turn, could impact effectiveness of the current WHO-recommended prepandemic H7 vaccines. PMID:23746830

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

    Christensen, Claus; Lauridsen, Jes B; Berezin, Vladimir; Bock, Elisabeth; Kiselyov, Vladislav V

    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...

  15. An approach for serotonin depletion in pigs: effects on serotonin receptor binding

    Ettrup, Anders; Kornum, Birgitte R; Weikop, Pia; Knudsen, Gitte M

    2011-01-01

    CPA caused average decreases in 5-HT concentrations of 61% ± 14% and 66% ± 16%, respectively, and a substantial loss of 5-HT immunostaining was seen throughout the brain. The serotonin depletion significantly increased 5-HT₄ receptor binding in nucleus accumbens, but did not alter 5-HT(1A) and 5-HT(2A...

  16. An approach for serotonin depletion in pigs: effects on serotonin receptor binding

    Ettrup, Anders; Kornum, Birgitte R; Weikop, Pia; Knudsen, Gitte M

    2011-01-01

    CPA caused average decreases in 5-HT concentrations of 61% ± 14% and 66% ± 16%, respectively, and a substantial loss of 5-HT immunostaining was seen throughout the brain. The serotonin depletion significantly increased 5-HT4 receptor binding in nucleus accumbens, but did not alter 5-HT(1A) and 5-HT(2A...

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

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

    1991-01-01

    The purified urokinase plasminogen activator receptor (u-PAR) was cleaved into two fragments by mild chymotrypsin treatment. The smaller fragment (apparent Mr 16,000) possessed the ligand-binding capability, as shown by chemical cross-linking analysis. This fragment constituted the NH2-terminal p...

  18. The effect of hyperthyroidism on opiate receptor binding and pain sensitivity

    This study was conducted to determine the effect of thyroid hormone on opiate receptor ligand-binding and pain sensitivity. Specific opiate receptor-binding was performed on brain homogenates of Swiss-Webster mice. There was a significant increase in 3H-naloxone-binding in thyroxine-fed subjects (hyperthyroid). Scatchard analysis revealed that the number of opiate receptors was increased in hyperthyroid mice (Bmax = 0.238 nM for hyperthyroid samples vs. 0.174 nM for controls). Binding affinity was unaffected (Kd = 1.54 nM for hyperthyroid and 1.58 nM for control samples). When mice were subjected to hotplate stimulation, the hyperthyroid mice were noted to be more sensitive as judged by pain aversion response latencies which were half that of control animals. After morphine administration, the hyperthyroid animals demonstrated a shorter duration of analgesia. These findings demonstrate that thyroxine increases opiate receptor number and native pain sensitivity but decreases the duration of analgesia from morphine

  19. Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    residue responded differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop...

  20. The effect of hyperthyroidism on opiate receptor binding and pain sensitivity

    Edmondson, E.A. (Baylor College of Medicine, Houston, TX (USA)); Bonnet, K.A.; Friedhoff, A.J. (New York Univ. School of Medicine, NY (USA))

    1990-01-01

    This study was conducted to determine the effect of thyroid hormone on opiate receptor ligand-binding and pain sensitivity. Specific opiate receptor-binding was performed on brain homogenates of Swiss-Webster mice. There was a significant increase in {sup 3}H-naloxone-binding in thyroxine-fed subjects (hyperthyroid). Scatchard analysis revealed that the number of opiate receptors was increased in hyperthyroid mice (Bmax = 0.238 nM for hyperthyroid samples vs. 0.174 nM for controls). Binding affinity was unaffected (Kd = 1.54 nM for hyperthyroid and 1.58 nM for control samples). When mice were subjected to hotplate stimulation, the hyperthyroid mice were noted to be more sensitive as judged by pain aversion response latencies which were half that of control animals. After morphine administration, the hyperthyroid animals demonstrated a shorter duration of analgesia. These findings demonstrate that thyroxine increases opiate receptor number and native pain sensitivity but decreases the duration of analgesia from morphine.

  1. Mutations in the hemagglutinin receptor-binding site can change the biological properties of an influenza virus.

    Naeve, C W; Hinshaw, V S; Webster, R G

    1984-01-01

    Avian influenza virus reassortants containing human influenza virus hemagglutinins do not replicate in ducks. Two mutations in the receptor-binding site of a human hemagglutinin at residues 226 and 228 allowed replication in ducks. The mutations resulted in a receptor-binding-site sequence identical to the known avian influenza virus sequences.

  2. The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases

    Pagh, Rasmus; Duus, Karen; Laursen, Inga; Hansen, Paul Robert; Mangor, Julie; Thielens, Nicole; Arlaud, Gérard J.; Kongerslev, Leif; Højrup, Peter; Houen, Gunnar

    2008-01-01

    The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL...... interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins...

  3. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

    Jenkins Jeremy L

    2001-10-01

    Full Text Available Abstract Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm aminopeptidase N (APN and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM, and unusually tight binding to the cadherin-like receptor (2.6 nM, which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research.

  4. The complex interplay between ligand binding and conformational structure of the folate binding protein (folate receptor)

    Holm, Jan; Bruun, Susanne Wrang; Hansen, Steen I.

    2015-01-01

    folate, probably due to shielding of binding sites between interacting hydrophobic patches. Titration with folate removes apo-monomers, favoring dissociation of self-associated apo-FBP into apo-monomers. Folate anchors to FBP through a network of hydrogen bonds and hydrophobic interactions, and the...... binding induces a conformational change with formation of hydrophilic and stable holo-FBP. Holo-FBP exhibits a ligand-mediated concentration-dependent self-association into multimers of great thermal and chemical stability due to strong intermolecular forces. Both ligand and FBP are thus protected against...

  5. Biological activities of binding site specific monoclonal antibodies to prolactin receptors of rabbit mammary gland

    The biological activity of three monoclonal antibodies (mAbs) against the rabbit mammary prolactin (PRL) receptor (M110, A82, and A917) were investigated using explants of rabbit mammary gland. The three mAbs which were all able to inhibit the binding of 125I-ovine prolactin to its receptor had different biological activities. Two mAbs (M110 and A82) were able to prevent the stimulating effect of PRL on casein synthesis when the molar ratio between the mAb and PRL was 100. One mAb (A917) was able to mimic the action of PRL on both casein and DNA ([3H]thymidine incorporation) synthesis, whereas the other two mAbs were without any stimulatory effect. For this stimulatory effect to be observed, bivalency of the antibody was essential, since monovalent fragments, which were able to inhibit PRL binding, had no agonistic activity. The ability of the mAbs to induce a down-regulation of receptors was also studied. These studies suggest that the binding domain of the receptor might be relatively complex, since only a part of this domain recognized by the antibody with PRL-like activity was able to induce hormonal action. Alternatively, only those antibodies able to microaggregate the receptors may possess PRL-like activity

  6. Defining the functional binding sites of interleukin 12 receptor β1 and interleukin 23 receptor to Janus kinases.

    Floss, Doreen M; Klöcker, Tobias; Schröder, Jutta; Lamertz, Larissa; Mrotzek, Simone; Strobl, Birgit; Hermanns, Heike; Scheller, Jürgen

    2016-07-15

    The interleukin (IL)-12-type cytokines IL-12 and IL-23 are involved in T-helper (Th) 1 and Th17 immunity, respectively. They share the IL-12 receptor β1 (IL-12Rβ1) as one component of their receptor signaling complexes, with IL-12Rβ2 as second receptor for IL-12 and IL-23R for IL-23 signal transduction. Stimulation with IL-12 and IL-23 results in activation of receptor-associated Janus kinases (Jak) and phosphorylation of STAT proteins in target cells. The Janus kinase tyrosine kinase (Tyk) 2 associates with IL-12Rβ1, whereas Jak2 binds to IL-23R and also to IL-12Rβ2. Receptor association of Jak2 is mediated by Box1 and Box2 motifs located within the intracellular domain of the receptor chains. Here we define the Box1 and Box2 motifs in IL-12Rβ1 and an unusual Jak2-binding site in IL-23R by the use of deletion and site-directed mutagenesis. Our data show that nonfunctional box motifs abolish IL-12- and IL-23-induced STAT3 phosphorylation and cytokine-dependent proliferation of Ba/F3 cells. Coimmunoprecipitation of Tyk2 by IL-12Rβ1 and Jak2 by IL‑23R supported these findings. In addition, our data demonstrate that association of Jak2 with IL-23R is mandatory for IL-12 and/or IL-23 signaling, whereas Tyk2 seems to be dispensable. PMID:27193299

  7. Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor: transfer of binding site to the CXCR3 receptor

    Rosenkilde, Mette M; Gerlach, Lars-Ole; Jakobsen, Janus S; Skerlj, Renato T; Bridger, Gary J; Schwartz, Thue W

    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......(262) (AspVI:23), and Glu(288) (GluVII:06) as the main interaction points for AMD3100. Molecular modeling suggests that one cyclam ring of AMD3100 interacts with Asp(171) in TM-IV, whereas the other ring is sandwiched between the carboxylic acid groups of Asp(262) and Glu(288) from TM-VI and -VII......, 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...

  8. Computational exploration of a protein receptor binding space with student proposed peptide ligands.

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

    2016-01-01

    Computational molecular docking is a fast and effective in silico method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The DockoMatic tutorial described herein provides a framework by which instructors can guide students through a drug screening exercise. Using receptor models derived from readily available protein crystal structures, docking programs have the ability to predict ligand binding properties, such as preferential binding orientations and binding affinities. The use of computational studies can significantly enhance complimentary wet chemical experimentation by providing insight into the important molecular interactions within the system of interest, as well as guide the design of new candidate ligands based on observed binding motifs and energetics. In this laboratory tutorial, the graphical user interface, DockoMatic, facilitates docking job submissions to the docking engine, AutoDock 4.2. The purpose of this exercise is to successfully dock a 17-amino acid peptide, α-conotoxin TxIA, to the acetylcholine binding protein from Aplysia californica-AChBP to determine the most stable binding configuration. Each student will then propose two specific amino acid substitutions of α-conotoxin TxIA to enhance peptide binding affinity, create the mutant in DockoMatic, and perform docking calculations to compare their results with the class. Students will also compare intermolecular forces, binding energy, and geometric orientation of their prepared analog to their initial α-conotoxin TxIA docking results. PMID:26537635

  9. The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor

    Kratochwil Nicole A

    2007-10-01

    Full Text Available Abstract Background Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone. Results By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore. Conclusion From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor

  10. The complex binding mode of the peptide hormone H2 relaxin to its receptor RXFP1.

    Sethi, Ashish; Bruell, Shoni; Patil, Nitin; Hossain, Mohammed Akhter; Scott, Daniel J; Petrie, Emma J; Bathgate, Ross A D; Gooley, Paul R

    2016-01-01

    H2 relaxin activates the relaxin family peptide receptor-1 (RXFP1), a class A G-protein coupled receptor, by a poorly understood mechanism. The ectodomain of RXFP1 comprises an N-terminal LDLa module, essential for activation, tethered to a leucine-rich repeat (LRR) domain by a 32-residue linker. H2 relaxin is hypothesized to bind with high affinity to the LRR domain enabling the LDLa module to bind and activate the transmembrane domain of RXFP1. Here we define a relaxin-binding site on the LDLa-LRR linker, essential for the high affinity of H2 relaxin for the ectodomain of RXFP1, and show that residues within the LDLa-LRR linker are critical for receptor activation. We propose H2 relaxin binds and stabilizes a helical conformation of the LDLa-LRR linker that positions residues of both the linker and the LDLa module to bind the transmembrane domain and activate RXFP1. PMID:27088579

  11. Intact brain cells: a novel model system for studying opioid receptor binding

    Rogers, N.F.; El-Fakahany, E.E.

    1985-07-29

    The use of a novel tissue preparation to study opioid receptor binding in viable, intact cells derived from whole brains of adult rats is presented. Mechanically dissociated and sieved cells, which were not homogenized at any stage of the experimental protocol, and iso-osmotic physiological buffer were used in these experiments. This system was adapted in order to avoid mechanical and chemical disruption of cell membranes, cytoskeletal ultrastructure or receptor topography by homogenization or by the use of nonphysiological buffers, and to mimic in vivo binding conditions as much as possible. Using (/sup 3/H)naloxone as the radioligand, the studies showed saturable and stereospecific high-affinity binding of this opioid antagonist in intact cells, which in turn showed consistently high viability. (/sup 3/H)Naloxone binding was also linear over a wide range of tissue concentrations. This technique provides a very promising model for future studies of the binding of opioids and of many other classes of drugs to brain tissue receptors in a more physiologically relevant system than those commonly used to date.

  12. Intact brain cells: a novel model system for studying opioid receptor binding

    The use of a novel tissue preparation to study opioid receptor binding in viable, intact cells derived from whole brains of adult rats is presented. Mechanically dissociated and sieved cells, which were not homogenized at any stage of the experimental protocol, and iso-osmotic physiological buffer were used in these experiments. This system was adapted in order to avoid mechanical and chemical disruption of cell membranes, cytoskeletal ultrastructure or receptor topography by homogenization or by the use of nonphysiological buffers, and to mimic in vivo binding conditions as much as possible. Using [3H]naloxone as the radioligand, the studies showed saturable and stereospecific high-affinity binding of this opioid antagonist in intact cells, which in turn showed consistently high viability. [3H]Naloxone binding was also linear over a wide range of tissue concentrations. This technique provides a very promising model for future studies of the binding of opioids and of many other classes of drugs to brain tissue receptors in a more physiologically relevant system than those commonly used to date

  13. Temperature dependence of estrogen binding: importance of a subzone in the ligand binding domain of a novel piscine estrogen receptor.

    Tan, N S; Frecer, V; Lam, T J; Ding, J L

    1999-11-11

    The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones beta1 and beta3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a approximately 3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37 degrees C, OaER requires approximately 80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37 degrees C to 25 degrees C or 20 degrees C resulted in a 4-fold increase in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25 degrees C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactivation capability. Amino acid replacements of OaER beta3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRalpha apo, RARgamma holo and ERalpha LBDs. Unliganded and 17beta-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone beta3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties. PMID:10559464

  14. Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1

    Ou, Xiuyuan; Góes, Luiz Gustavo Bentim; Osborne, Christina; Castano, Anna; Holmes, Kathryn V.

    2015-01-01

    ABSTRACT Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (β-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the β-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several α-CoVs use the downstream C domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human β-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (MAbs) to the ectodomain of HKU1 spike protein. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike MAbs recognized epitopes in the C domain between amino acids 535 and 673, indicating that this region is immunodominant. Two of the MAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Preincubation of HTBE cells with a truncated HKU1 S protein that includes the C domain blocked infection with HKU1 virus, but preincubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C domain, where the spike proteins of α-CoVs and β-CoVs in groups B and C bind to their specific receptor proteins. Thus, two β-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins. IMPORTANCE Mouse hepatitis virus, a β-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another β-CoV in group A, uses the NTD to bind to its sialic-acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory β-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the

  15. Label-free impedimetric biosensor for thrombin using the thrombin-binding aptamer as receptor

    Frense, D.; Kang, S.; Schieke, K.; Reich, P.; Barthel, A.; Pliquett, U.; Nacke, T.; Brian, C.; Beckmann, D.

    2013-04-01

    This study presents the further establishment of impedimetric biosensors with aptamers as receptors. Aptamers are short single-stranded oligonucleotides which bind analytes with a specific region of their 3D structure. Electrical impedance spectroscopy is a sensitive method for analyzing changes on the electrode surface, e.g. caused by receptor-ligand-interactions. Fast and inexpensive prototyping of electrodes on the basis of commercially available compact discs having a 24 carat gold reflective layer was investigated. Electrode structures (CDtrodes [1]) in the range from few millimetres down to 100 microns were realized. The well-studied thrombin-binding aptamer (TBA) was used as receptor for characterizing these micro- and macro-electrodes. The impedance signal showed a linear correlation for concentrations of thrombin between 1.0 nM to 100 nM. This range corresponds well with most of the references and may be useful for the point-of-care testing (POCT).

  16. Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

    Molinari, A M; Abbondanza, C; Armetta, I; Medici, N; Minucci, S; Moncharmont, B; Nigro, V; Puca, G A

    1991-05-15

    The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity. PMID:1709742

  17. Developmental Profile of the Aberrant Dopamine D2 Receptor Response in Striatal Cholinergic Interneurons in DYT1 Dystonia

    Giuseppe Sciamanna; Annalisa Tassone; Giuseppina Martella; Georgia Mandolesi; Francesca Puglisi; Dario Cuomo; Grazia Madeo; Giulia Ponterio; David George Standaert; Paola Bonsi; Antonio Pisani

    2011-01-01

    BACKGROUND: DYT1 dystonia, a severe form of genetically determined human dystonia, exhibits reduced penetrance among carriers and begins usually during adolescence. The reasons for such age dependence and variability remain unclear. METHODS AND RESULTS: We characterized the alterations in D2 dopamine receptor (D2R) signalling in striatal cholinergic interneurons at different ages in mice overexpressing human mutant torsinA (hMT). An abnormal excitatory response to the D2R agonist quinpirole w...

  18. Neuroplastin-55 binds to and signals through the fibroblast growth factor receptor

    Owczarek, Sylwia; Kiryushko, Darya; Hald Larsen, Marianne;

    2010-01-01

    3), whereas Np55 lacks the Ig1 module. Of these two isoforms, only Np65 is involved in homophilic interactions resulting in cell adhesion, whereas the role of Np55 is poorly understood. The present study reports for the first time the crystal structure of the ectodomain of Np55 at 1.95-A resolution...... and demonstrates that Np55 binds to and activates the fibroblast growth factor receptor 1 (FGFR1). Furthermore, we identify a sequence motif in the Ig2 module of Np55 interacting with FGFR1 and show that a synthetic peptide encompassing this motif, termed narpin, binds to and activates FGFR1. We show...... Np55-induced signaling may be involved in synaptic plasticity in vivo. Owczarek, S., Kiryushko, D., Larsen, M. H., Kastrup, J. S., Gajhede, M., Sandi, C., Berezin, V., Bock, E., Soroka, V. Neuroplastin-55 binds to and signals through the fibroblast growth factor receptor....

  19. Decreased frontal serotonin 5-HT2a receptor binding index in deliberate self-harm patients

    Studies of serotonin metabolites in body fluids in attempted suicide patients and of post-mortem brain tissue of suicide victims have demonstrated the involvement of the serotonergic neurotransmission system in the pathogenesis of suicidal behaviour. Recently developed neuroimaging techniques offer the unique possibility of investigating in vivo the functional characteristics of this system. In this study the 5-HT2a receptor population of patients who had recently attempted suicide was studied by means of the highly specific radio-iodinated 5-HT2a receptor antagonist 4-amino-N-[1-[3-(4-fluorophenoxy)propyl]-4-methyl-4-piperidinyl] -5-iodo-2-methox ybenzamide or 123I-5-I-R91150. Nine patients who had recently (1-7 days) attempted suicide and 12 age-matched healthy controls received an intravenous injection of 185 MBq 123I-5-I-R91150 and were scanned with high-resolution brain single-photon emission tomography (SPET). Stereotactic realigned images were analysed semi-quantitatively using predefined volumes of interest. Serotonin binding capacity was expressed as the ratio of specific to non-specific activity. The cerebellum was used as a measure of non-specific activity. An age-dependent 5-HT2a binding index was found, in agreement with previous literature. Deliberate self-harm patients had a significantly reduced mean frontal binding index after correction for age (P=0.002) when compared with controls. The reduction was more pronounced among deliberate self-injury patients (DSI) (P2a serotonin receptor system in attempted suicide patients who are free of drugs influencing the serotonergic system shows in vivo evidence of a decreased frontal binding index of the 5-HT2a receptor, indicating a decrease in the number and/or in the binding affinity of 5-HT2a receptors. (orig.)

  20. Ondansetron and Granisetron Binding Orientation in the 5-HT3 Receptor Determined by Unnatural Amino Acid Mutagenesis

    Duffy, Noah H.; Lester, Henry A.; Dougherty, Dennis A.

    2012-01-01

    The serotonin type 3 receptor (5-HT3R) is a ligand-gated ion channel that mediates fast synaptic transmission in the central and peripheral nervous systems. The 5-HT3R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT3A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket. PMID:22873819

  1. Synthesis and binding characteristics of [(3)H]neuromedin N, a NTS2 receptor ligand.

    Tóth, Fanni; Mallareddy, Jayapal Reddy; Tourwé, Dirk; Lipkowski, Andrzej W; Bujalska-Zadrozny, Magdalena; Benyhe, Sándor; Ballet, Steven; Tóth, Géza; Kleczkowska, Patrycja

    2016-06-01

    Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors. The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [(3)H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454nM in rat brain and 425nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8±30.18nM, while the Bmax value corresponded to 3.8±0.2pmol/mg protein in rat brain membranes. The specific binding of [(3)H]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na(+) ions was observed on the binding of [(3)H]NN, resulting in an IC50 of 150.6mM. In [(35)S]GTPγS binding experiments the Emax value was 112.3±1.4% in rat brain and 112.9±2.4% in rat spinal cord membranes and EC50 values of 0.7nM and 0.79nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [(3)H]NN labels NTS2 receptors in rat brain membranes. PMID:26707235

  2. Immunohistology of oestrogen receptor and D5 antigen in breast cancer: correlation with oestrogen receptor content of adjacent cryostat sections assayed by radioligand binding and enzyme immunoassay.

    Giri, D. D.; Dangerfield, V J; Lonsdale, R; Rogers, K.; Underwood, J C

    1987-01-01

    Two monoclonal antibodies recognising epitopes associated with oestrogen receptor protein were evaluated against the assayable soluble oestrogen receptor concentration in a series of 149 breast carcinomas. One antibody (anti-ER) recognises the hormone binding unit of oestrogen receptor and gives nuclear staining; the other antibody (anti-D5) was raised to a component of soluble oestrogen receptor and gives cytoplasmic staining. To minimise variations attributable to tumour heterogeneity and s...

  3. A 67 kDa non-hormone binding estradiol receptor is present in human mammary cancers.

    Castoria, G; Migliaccio, A; Bilancio, A; Pagano, M; Abbondanza, C; Auricchio, F

    1996-03-01

    The presence of large amounts of a 67 kDa estradiol receptor that does not bind hormone was observed in 8 to 37 human mammary tumors (34 malignant and 3 benign). This form of receptor was detected by its conversion to hormone binding receptor by an endogenous tyrosine kinase in vitro. All 8 tumors were malignant. In these, the incubation of cytosol with ATP was seen to cause a 1- to 5-fold increase in estradiol-specific binding sites. These sites bound estradiol with physiological affinity, and their appearance was associated with tyrosine phosphorylation of estradiol receptor. The enzyme converting the non-hormone binding receptor into the hormone binding receptor is largely present in cytosol and scarce in membranes. It has been extensively purified. It is a 67 kDa protein under denaturating conditions, binds calmodulin-Sepharose in a Ca2+-dependent manner, is stimulated by Ca2+ and calmodulin, phosphorylates exogenous actin, is activated by the estradiol-receptor complex. The enzyme interacts with antibodies directed against the carboxy-terminal and catalytic domains of c-src. Therefore, it is a putative new member of the large c-src-related kinase family. Human mammary cancers with significant amounts of 67 kDa non-hormone binding receptor show relatively low levels of hormone binding estradiol receptor. The presence of non-hormone binding receptor that can be activated by in vitro tyrosine phosphorylation suggests that functional interaction of estradiol receptor with tyrosine kinases is altered in malignant tumors and has bearing on loss of hormone dependence and progression of the mammary cancer malignancy. PMID:8598306

  4. Structure of the ligand-binding domain of the EphB2 receptor at 2 Å resolution

    The crystal structure of the ligand-binding domain of a receptor tyrosine kinase EphB2, an important mediator of cell-cell communication, has been determined at a resolution of 2 Å. The structure confirms the induced-fit mechanism for the binding of ligands to EphB receptors. Eph tyrosine kinase receptors, the largest group of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell–cell communication regulating cell attachment, shape and mobility. Recently, several Eph receptors and ephrins have also been found to play important roles in the progression of cancer. Structural and biophysical studies have established detailed information on the binding and recognition of Eph receptors and ephrins. The initial high-affinity binding of Eph receptors to ephrin occurs through the penetration of an extended G–H loop of the ligand into a hydrophobic channel on the surface of the receptor. Consequently, the G–H loop-binding channel of Eph receptors is the main target in the search for Eph antagonists that could be used in the development of anticancer drugs and several peptides have been shown to specifically bind Eph receptors and compete with the cognate ephrin ligands. However, the molecular details of the conformational changes upon Eph/ephrin binding have remained speculative, since two of the loops were unstructured in the original model of the free EphB2 structure and their conformational changes upon ligand binding could consequently not be analyzed in detail. In this study, the X-ray structure of unbound EphB2 is reported at a considerably higher 2 Å resolution, the conformational changes that the important receptor loops undergo upon ligand binding are described and the consequences that these findings have for the development of Eph antagonists are discussed

  5. 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

    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.

  6. Ring size in cyclic endomorphin-2 analogs modulates receptor binding affinity and selectivity.

    Piekielna, Justyna; Kluczyk, Alicja; Gentilucci, Luca; Cerlesi, Maria Camilla; Calo', Girolamo; Tomböly, Csaba; Łapiński, Krzysztof; Janecki, Tomasz; Janecka, Anna

    2015-06-01

    The study reports the solid-phase synthesis and biological evaluation of a series of new side chain-to-side chain cyclized opioid peptide analogs of the general structure Tyr-[D-Xaa-Phe-Phe-Asp]NH2, where Xaa = Lys (1), Orn (2), Dab (3), or Dap (4) (Dab = 2,4-diaminobutyric acid, Dap = 2,3-diaminopropionic acid), containing 17- to 14-membered rings. The influence of the ring size on binding to the MOP, DOP and KOP opioid receptors was studied. In general, the reduction of the size of the macrocyclic ring increased the selectivity for the MOP receptor. The cyclopeptide incorporating Xaa = Lys displayed subnanomolar MOP affinity but modest selectivity over the KOP receptor, while the analog with the Orn residue showed increased affinity and selectivity for MOP. The analog with Dab was a weak MOP agonist and did not bind to the other two opioid receptors. Finally, the peptide with Xaa = Dap was completely MOP receptor-selective with subnanomolar affinity. Interestingly, the deletion of one Phe residue from 1 led to the 14-membered Tyr-c[D-Lys-Phe-Asp]NH2 (5), a potent and selective MOP receptor ligand. The in vitro potencies of the new analogs were determined in a calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. A good correlation between binding and the functional test results was observed. The influence of the ring size, solid support and the N-terminal protecting group on the formation of cyclodimers was studied. PMID:25948019

  7. Binding of 125I-human growth hormone to specific receptors in human cultured lymphocytes

    The interaction of human growth hormone with human lymphocytes from an established culture (IM-9) was studied using 125I- human growth hormone. The binding of 125I-human growth hormone was rapid; with human growth hormone at 0.1 nM a steady state was observed in 90 min at 300. Bound labeled human growth hormone was dissociated rapidly by addition of excess unlabeled human growth hormone. Binding of 125I-human growth hormone to cultured lymphocytes was relatively insensitive to alterations in the pH and in the concentrations of Ca2+, Mg2+, EDTA. At 800 there was very little degradation of labeled human growth hormone or of the specific receptor sites. Tryptic digestion destroyed the capacity of cells to bind human growth hormone. The IM-9 cells bound all human growth hormone preparations but not unrelated hormones or nonprimate growth hormones. The binding of 125I-human growth hormone was inhibited 10 to 14 percent with 1 to 2 ng per ml of unlabeled human growth hormone and 50 percent with 30 to 40 ng per ml, well within the range of hormone concentrations in vivo. Analysis of steady state data revealed a single order of binding sites with an affinity constant of 1.3 x 109 M-1 and about 4000 binding sites per cell. Numerous human growth hormone preparations were assayed by use of this receptor system as well as by immunoassay and by bioassay in vivo. The po

  8. Computational study of the binding modes of caffeine to the adenosine A2A receptor.

    Liu, Yuli; Burger, Steven K; Ayers, Paul W; Vöhringer-Martinez, Esteban

    2011-12-01

    Using the recently solved crystal structure of the human adenosine A(2A) receptor, we applied MM/PBSA to compare the binding modes of caffeine with those of the high-affinity selective antagonist ZM241385. MD simulations were performed in the environment of the lipid membrane bilayer. Four low-energy binding modes of caffeine-A(2A) were found, all of which had similar energies. Assuming an equal contribution of each binding mode of caffeine, the computed binding free energy difference between caffeine and ZM241385 is -2.4 kcal/mol, which compares favorably with the experimental value, -3.6 kcal/mol. The configurational entropy contribution of -0.9 kcal/mol from multiple binding modes of caffeine helps explain how a small molecule like caffeine can compete with a significantly larger molecule, ZM241385, which can form many more interactions with the receptor. We also performed residue-wise energy decomposition and found that Phe168, Leu249, and Ile274 contribute most significantly to the binding modes of caffeine and ZM241385. PMID:21970461

  9. Different binding of stimulatory-type and blocking-type TSH receptor antibody with guinea-pig testis membrane.

    Inui, T; Ochi, Y; Hachiya, T; Chen, W; Nakajima, Y; Kajita, Y; Ogura, H

    1991-11-01

    A receptor assay using [125I]bTSH-binding to guinea-pig testis membrane was developed. Unlabelled hCG and FSH inhibited [125I]bTSH binding. In patients with Graves' disease and in untreated hyperthyroid patients, almost all long-acting thyroid stimulators and thyroid-stimulating antibodies, respectively did not inhibit [125I]bTSH binding, which on the other hand was inhibited by thyroid stimulation blocking antibodies in patients with primary hypothyroidism. When the inhibitory effect on the binding of [125I]hCG and 125I-synthetic alpha-subunit peptide (alpha 26-46) of hCG to testis membrane was examined, bTSH resulted in a significant inhibition. However, all three kinds of TSH receptor antibodies had no inhibitory effect. This study demonstrated 1. interaction of alpha-subunit of TSH and hCG with the testicular receptor; 2. binding of thyroid stimulation-blocking antibody and lack of binding of thyroid-stimulating antibody to the testicular TSH receptor in spite of binding of these TSH receptor antibodies to the thyroidal TSH receptor, and 3. lack of binding of thyroid-stimulating antibody and thyroid stimulation-blocking antibody to the testicular gonadotropin receptor. PMID:1684686

  10. Localization and synthesis of an insulin-binding region on human insulin receptor

    Seven regions of the alpha subunit of human insulin receptor (HIR) were synthesized and examined for their ability to bind radioiodinated insulin. A peptide representing one of these regions (namely, residues alpha 655-670) exhibited a specific binding activity for insulin. In quantitative radiometric titrations, the binding curves of 125I-labeled insulin to adsorbents of peptide alpha 655-670 and of purified placental membrane were similar or superimposable. The binding of radioiodinated insulin to peptide or to membrane adsorbents was completely inhibited by unlabeled insulin, and the inhibition curves indicated that the peptide and the membrane on the adsorbents had similar affinities. Synthetic peptides that were shorter (peptide alpha 661-670) or longer (peptide alpha 651-670) than the region alpha 655-670 exhibited lower insulin-binding activity. It was concluded that an insulin-binding region in the HIR alpha subunit resides within residues alpha 655-670. The results do not rule out the possibility that other regions of the alpha subunit may also participate in binding of HIR to insulin, with the region described here forming a face within a larger binding site

  11. Localization and synthesis of an insulin-binding region on human insulin receptor

    Nakamura, S.; Sakata, S.; Atassi, M.Z. (Baylor College of Medicine, Houston, TX (USA))

    1990-04-01

    Seven regions of the alpha subunit of human insulin receptor (HIR) were synthesized and examined for their ability to bind radioiodinated insulin. A peptide representing one of these regions (namely, residues alpha 655-670) exhibited a specific binding activity for insulin. In quantitative radiometric titrations, the binding curves of {sup 125}I-labeled insulin to adsorbents of peptide alpha 655-670 and of purified placental membrane were similar or superimposable. The binding of radioiodinated insulin to peptide or to membrane adsorbents was completely inhibited by unlabeled insulin, and the inhibition curves indicated that the peptide and the membrane on the adsorbents had similar affinities. Synthetic peptides that were shorter (peptide alpha 661-670) or longer (peptide alpha 651-670) than the region alpha 655-670 exhibited lower insulin-binding activity. It was concluded that an insulin-binding region in the HIR alpha subunit resides within residues alpha 655-670. The results do not rule out the possibility that other regions of the alpha subunit may also participate in binding of HIR to insulin, with the region described here forming a face within a larger binding site.

  12. In Silico Investigation of the Neurotensin Receptor 1 Binding Site: Overlapping Binding Modes for Small Molecule Antagonists and the Endogenous Peptide Agonist

    Lückmann, Michael; Holst, Birgitte; Schwartz, Thue W.; Frimurer, Thomas Michael

    2015-01-01

    The neurotensin receptor 1 (NTSR1) belongs to the family of 7TM, G protein-coupled receptors, and is activated by the 13-amino-acid peptide neurotensin (NTS) that has been shown to play important roles in neurological disorders and the promotion of cancer cells. Recently, a high-resolution x......-ray crystal structure of NTSR1 in complex with NTS8–13 has been determined, providing novel insights into peptide ligand recognition by 7TM receptors. SR48692, a potent and selective small molecule antagonist has previously been used extensively as a tool compound to study NTSR1 receptor signaling properties....... To investigate the binding mode of SR48692 and other small molecule compounds to NTSR1, we applied an Automated Ligand-guided Backbone Ensemble Receptor Optimization protocol (ALiBERO), taking receptor flexibility and ligand knowledge into account. Structurally overlapping binding poses for SR48692...

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

    Sun, Hui; KAWAGUCHI, RIKI

    2011-01-01

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

  14. Optical Aberrations and Wavefront

    Nihat Polat

    2014-08-01

    Full Text Available The deviation of light to create normal retinal image in the optical system is called aberration. Aberrations are divided two subgroup: low-order aberrations (defocus: spherical and cylindrical refractive errors and high-order aberrations (coma, spherical, trefoil, tetrafoil, quadrifoil, pentafoil, secondary astigmatism. Aberrations increase with aging. Spherical aberrations are compensated by positive corneal and negative lenticular spherical aberrations in youth. Total aberrations are elevated by positive corneal and positive lenticular spherical aberrations in elderly. In this study, we aimed to analyze the basic terms regarding optic aberrations which have gained significance recently. (Turk J Ophthalmol 2014; 44: 306-11

  15. A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens

    J. Veldscholte (Jos); C. Ris-Stalpers (Carolyn); G.G.J.M. Kuiper (George); G.W. Jenster (Guido); C.A. Berrevoets (Cor); H.J.H.M. Claassen (Eric); H.C.J. van Rooij (Henri); J. Trapman (Jan); A.O. Brinkmann (Albert); E. Mulder (Eppo)

    1990-01-01

    markdownabstractAbstract INCaP prostate tumor cells contain an abnormal androgen receptor system. Progestagens, estradiol and anti-androgens can compete with androgens for binding to the androgen receptor and can stimulate both cell growth and excretion of prostate specific acid phosphatase. We ha

  16. Structural Studies of Nicotinic Acetylcholine Receptors: Using Acetylcholine-Binding Protein as a Structural Surrogate.

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette S; Balle, Thomas

    2016-06-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in the control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for the development of drugs against a number of mental health disorders and for marketed smoking cessation aids. Unfortunately, drug discovery has been hampered by difficulties in obtaining sufficiently selective compounds. Together with functional complexity of the receptors, this has made it difficult to obtain drugs with sufficiently high-target to off-target affinity ratios. The recent and ongoing progress in structural studies holds promise to help understand structure-function relationships of nAChR drugs at the atomic level. This will undoubtedly lead to the design of more efficient drugs with fewer side effects. As a high-resolution structure of a nAChR is yet to be determined, structural studies are to a large extent based on acetylcholine-binding proteins (AChBPs) that despite low overall sequence identity display a high degree of conservation of overall structure and amino acids at the ligand-binding site. Further, AChBPs reproduce relative binding affinities of ligands at nAChRs. Over the past decade, AChBPs have been used extensively as models for nAChRs and have aided the understanding of drug receptor interactions at nAChRs significantly. PMID:26572235

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

    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...

  18. Electrostatics and Intrinsic Disorder Drive Translocon Binding of the SRP Receptor FtsY.

    Lakomek, Nils-Alexander; Draycheva, Albena; Bornemann, Thomas; Wintermeyer, Wolfgang

    2016-08-01

    Integral membrane proteins in bacteria are co-translationally targeted to the SecYEG translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N-terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C-terminal membrane targeting sequence. The central A domain binds to the translocon non-specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome-bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane. PMID:27346853

  19. Insulin binding changes the interface region between α subunits of the insulin receptor

    The homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) was used to probe the interface region between the two α subunits of the α2β2 human insulin receptor. The two α subunits formed a covalent dimer when affinity-purified receptor or membrane-bound receptor was reacted with DSS. The α2 species was detected on protein blots from SDS gels using an anti-α-subunit antibody or 125I-concanavalin A. Alternatively, iodinated receptor was reacted with DSS and the α2 species measured directly in an SDS gel. As shown by all three assay systems, more α2 was formed when insulin was bound to receptor than when insulin was absent. These data indicate that the conformational change which occurs in the α subunit response to insulin binding results in a change in the α-α interaction within the receptor complex. The results are consistent with a kinase activation mechanism involving communication between the two αβ receptor halves

  20. Intracellular binding site kinetics of 201 Tl binding compared to β-adrenergic analog receptors in dog myocardium

    It has been demonstrated with the multiple indicator dilution technique (MID) in an isolated dog heart preparation, that the permeation of thallium ions across the sarcolemma is about ten times larger compared to potassium ions (cellular permeability surface area product PSM 8.90 +- 4.60 vs. 0.65 +- 0.46 ml/min gsup(-1)). Similarly, the intracellular (IC) distribution space of Tlsup(+) is larger compared to that of Ksup(+). These properties may explain in part the rapid and large extraction of Tl in the myocardium. To explain the slow washout rate of Tl from the myocardium (T 1/2>600 sec determined with an on-line residue detection) we proposed a temporary binding of Tl to an IC protein. In experiments the permeation properties of 201 Tl were compared to 125 I-cyanopinodolol (I-CP) and 131 I metabenzylquanidin (I-MBG) by means of MID. The latter two substances act at the β-adrenergic receptor site. Both substances have a lower capillary permeability surface area product PSC of 0.43 +- 0.37 ml/min gsup(-1) compared to that of 201 Tl (1.37 +- 0.49 ml/min gsup(-1)). I-CP and I-MBG are sequestered extracellularly in contrast to Tl, which permeates intracellularly. However, the relation between time and instantaneous extraction during a single bolus passage of 201 Tl is very comparable to that of those receptor substances suggesting also a receptor-type kinetics for Tl with intracellular binding which may elucidate its prolonged washout. (Author)

  1. Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

    Yang, Hua; Chen, Li-Mei; Carney, Paul J.; Donis, Ruben O.; Stevens, James (CDC)

    2012-02-21

    Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type ({alpha}2-3) receptor binding profile, with only moderate binding to human-type ({alpha}2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.

  2. Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway

    Varghese, Binny V.; Koohestani, Faezeh; McWilliams, Michelle; Colvin, Arlene; Gunewardena, Sumedha; Kinsey, William H.; Nowak, Romana A.; Nothnick, Warren B.; Chennathukuzhi, Vargheese M.

    2013-01-01

    Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase–protein kinase B/AKT–mammalian target of rapamycin (PI3K/AKT–mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K–AKT–mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids. PMID:23284171

  3. A receptor-binding region in Escherichia coli alpha-haemolysin.

    Cortajarena, Aitziber L; Goni, Félix M; Ostolaza, Helena

    2003-05-23

    Escherichia coli alpha-hemolysin (HlyA) is a 107-kDa protein toxin with a wide range of mammalian target cells. Previous work has shown that glycophorin is a specific receptor for HlyA in red blood cells (Cortajarena, A. L., Goñi, F. M., and Ostolaza, H. (2001) J. Biol. Chem. 276, 12513-12519). The present study was aimed at identifying the glycophorin-binding region in the toxin. Data in the literature pointed to a short amino acid sequence near the C terminus as a putative receptor-binding domain. Previous sequence analyses of several homologous toxins that belong, like HlyA, to the so-called RTX toxin family revealed a conserved region that corresponded to residues 914-936 of HlyA. We therefore prepared a deletion mutant lacking these residues (HlyA Delta 914-936) and found that its hemolytic activity was decreased by 10,000-fold with respect to the wild type. This deletion mutant was virtually unable to bind human and horse red blood cells or to bind pure glycophorin in an affinity column. The peptide Trp914-Arg936 had no lytic activity of its own, but it could bind glycophorin reconstituted in lipid vesicles. Moreover, the peptide Trp914-Arg936 protected red blood cells from hemolysis induced by wild type HlyA. It was concluded that amino acid residues 914-936 constitute a major receptor-binding region in alpha-hemolysin. PMID:12582172

  4. Localization and synthesis of the hormone-binding regions of the human thyrotropin receptor.

    Atassi, M Z; Manshouri, T; Sakata, S.

    1991-01-01

    Two regions of human thyrotropin (thyroid-stimulating hormone, TSH) receptor (TSHR) (residues 12-44 and 308-364) were selected on the basis that they exhibit no sequence resemblance to luteinizing hormone/chorionic gonadotropin receptor. Five synthetic overlapping peptides (12-30, 24-44, 308-328, 324-344, and 339-364) were studied for their ability to bind 125I-labeled human TSH (hTSH), its isolated alpha and beta subunits, bovine TSH, ovine TSH, human luteinizing hormone, and human follicle-...

  5. Brain serotonin 2A receptor binding: Relations to body mass index, tobacco and alcohol use

    Erritzoe, D.; Frokjaer, V. G.; Haugbol, S.;

    2009-01-01

    to increased food and alcohol intake, and conversely, stimulation of the serotonergic system induces weight reduction and decreased food/alcohol intake as well as tobacco smoking. To investigate whether body weight, alcohol intake and tobacco smoking were related to the regulation of the cerebral serotonin 2A...... receptor (5-HT(2A)) in humans, we tested in 136 healthy human subjects if body mass index (BMI), degree of alcohol consumption and tobacco smoking was associated to the cerebral in vivo 5-HT(2A) receptor binding as measured with (18)F-altanserin PET. The subjects' BMI's ranged from 18.4 to 42.8 (25...

  6. Characterization of 5-HT1D receptor binding sites in post-mortem human brain cortex.

    Martial, J; de Montigny, C; Cecyre, D; Quirion, R

    1991-01-01

    The present study provides further evidence for the presence of serotonin1D (5-HT1D) receptors in post-mortem human brain. Receptor binding parameters in temporal cortex homogenates were assessed using [3H]5-HT in the presence of 100 nM 8-OH-DPAT, 1 microM propranolol and 1 microM mesulergine to prevent labelling of the 5-HT1A, 5-HT1B and 5-HT1C sites, respectively. Under these conditions, [3H]5-HT apparently bound to a class of high affinity (Kd = 5.0 +/- 1.0 nM) low capacity (Bmax = 96 +/- ...

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

    Tony Velkov

    2013-01-01

    Full Text Available Fatty acid binding proteins (FABPs act as intracellular shuttles for fatty acids as well as lipophilic xenobiotics to the nucleus, where these ligands are released to a group of nuclear receptors called the peroxisome proliferator activated receptors (PPARs. PPAR mediated gene activation is ultimately involved in maintenance of cellular homeostasis through the transcriptional regulation of metabolic enzymes and transporters that target the activating ligand. Here we show that liver- (L- FABP displays a high binding affinity for PPAR subtype selective drugs. NMR chemical shift perturbation mapping and proteolytic protection experiments show that the binding of the PPAR subtype selective drugs produces conformational changes that stabilize the portal region of L-FABP. NMR chemical shift perturbation studies also revealed that L-FABP can form a complex with the PPAR ligand binding domain (LBD of PPARα. This protein-protein interaction may represent a mechanism for facilitating the activation of PPAR transcriptional activity via the direct channeling of ligands between the binding pocket of L-FABP and the PPARαLBD. The role of L-FABP in the delivery of ligands directly to PPARα via this channeling mechanism has important implications for regulatory pathways that mediate xenobiotic responses and host protection in tissues such as the small intestine and the liver where L-FABP is highly expressed.

  8. Receptor-like function of heparin in the binding and uptake of neutral lipids

    Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids 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, the authors 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 and 125I-labeled triglyceride lipase. 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. They 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 generalized pathway for transport of neutral lipids into endothelial and other cells

  9. Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.

    Wu, Qing-Ping; Zhang, Lei; Shao, Xiao-Xia; Wang, Jia-Hui; Gao, Yu; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-04-01

    Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1. PMID:26767372

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

    Knollman, Paul E.; Conn, P. Michael

    2008-01-01

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

  11. Brain serotonin 4 receptor binding is associated with the cortisol awakening response

    Jakobsen, Gustav R; Fisher, Patrick M; Dyssegaard, Agnete;

    2016-01-01

    Serotonin signalling is considered critical for an appropriate and dynamic adaptation to stress. Previously, we have shown that prefrontal serotonin transporter (SERT) binding is positively associated with the cortisol awakening response (CAR) (Frokjaer et al., 2013), which is an index of...... hypothalamic-pituitary-adrenal (HPA)-axis output dynamics. Here, we investigated in healthy individuals if cerebral serotonin 4 receptor (5-HT4r) binding, reported to be a proxy for serotonin levels, is associated with CAR. Thirty healthy volunteers (25 males, age range 20-56 years) underwent 5-HT4r PET...

  12. GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

    Yasumoto, Hiroaki; Meng, Lingjun; Lin, Tao; Zhu, Qubo; Tsai, Robert Y.L.

    2007-01-01

    Guanine-nucleotide binding protein 3-like (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1p-null yeasts. To determine the unique function of GNL3L, we identified estrogen receptor-related protein-γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The i...

  13. Characterization of Genomic Vitamin D Receptor Binding Sites through Chromatin Looping and Opening

    Seuter, Sabine; Neme, Antonio; Carlberg, Carsten

    2014-01-01

    The vitamin D receptor (VDR) is a transcription factor that mediates the genomic effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Genome-wide there are several thousand binding sites and hundreds of primary 1,25(OH)2D3 target genes, but their functional relation is largely elusive. In this study, we used ChIA-PET data of the transcription factor CTCF in combination with VDR ChIP-seq data, in order to map chromatin domains containing VDR binding sites. In total, we found 1,599 such VDR cont...

  14. Differential binding of prohibitin-2 to estrogen receptor α and to drug-resistant ERα mutants

    Endocrine resistance is one of the most challenging problems in estrogen receptor alpha (ERα)-positive breast cancer. The transcriptional activity of ERα is controlled by several coregulators, including prohibitin-2 (PHB2). Because of its ability to repress the transcriptional activity of activated ERα, PHB2 is a promising antiproliferative agent. In this study, were analyzed the interaction of PHB2 with ERα and three mutants (Y537S, D538G, and E380Q) that are frequently associated with a lack of sensitivity to hormonal treatments, to help advance novel drug discovery. PHB2 bound to ERα wild-type (WT), Y537S, and D538G, but did not bind to E380Q. The binding thermodynamics of Y537S and D538G to PHB2 were favorably altered entropically compared with those of WT to PHB2. Our results show that PHB2 binds to the ligand binding domain of ERα with a conformational change in the helix 12 of ERα. - Highlights: • Molten globule-likeness of an ERα repressor Prohibitin-2 (PHB2) is identified. • The thermodynamics is validated for the interaction between ERα and PHB2. • PHB2 binds to Y537S and D538G mutants of ERα commonly found in breast cancer. • ERα WT and mutants showed different thermodynamic parameters in the binding to PHB2. • ERα binds to PHB2 with conformational change involving packing of helix 12

  15. Differential binding of prohibitin-2 to estrogen receptor α and to drug-resistant ERα mutants

    Chigira, Takeru, E-mail: 8120661875@mail.ecc.u-tokyo.ac.jp [Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Nagatoishi, Satoru, E-mail: nagatoishi@bioeng.t.u-tokyo.ac.jp [Department of Bioengineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan); Tsumoto, Kouhei, E-mail: tsumoto@bioeng.t.u-tokyo.ac.jp [Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Bioengineering, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan)

    2015-08-07

    Endocrine resistance is one of the most challenging problems in estrogen receptor alpha (ERα)-positive breast cancer. The transcriptional activity of ERα is controlled by several coregulators, including prohibitin-2 (PHB2). Because of its ability to repress the transcriptional activity of activated ERα, PHB2 is a promising antiproliferative agent. In this study, were analyzed the interaction of PHB2 with ERα and three mutants (Y537S, D538G, and E380Q) that are frequently associated with a lack of sensitivity to hormonal treatments, to help advance novel drug discovery. PHB2 bound to ERα wild-type (WT), Y537S, and D538G, but did not bind to E380Q. The binding thermodynamics of Y537S and D538G to PHB2 were favorably altered entropically compared with those of WT to PHB2. Our results show that PHB2 binds to the ligand binding domain of ERα with a conformational change in the helix 12 of ERα. - Highlights: • Molten globule-likeness of an ERα repressor Prohibitin-2 (PHB2) is identified. • The thermodynamics is validated for the interaction between ERα and PHB2. • PHB2 binds to Y537S and D538G mutants of ERα commonly found in breast cancer. • ERα WT and mutants showed different thermodynamic parameters in the binding to PHB2. • ERα binds to PHB2 with conformational change involving packing of helix 12.

  16. Muscarinic receptor binding increases in anterior thalamus and cingulate cortex during discriminative avoidance learning

    Training-induced neuronal activity develops in the mammalian limbic system during discriminative avoidance conditioning. This study explores behaviorally relevant changes in muscarinic ACh receptor binding in 52 rabbits that were trained to one of five stages of conditioned response acquisition. Sixteen naive and 10 animals yoked to criterion performance served as control cases. Upon reaching a particular stage of training, the brains were removed and autoradiographically assayed for 3H-oxotremorine-M binding with 50 nM pirenzepine (OxO-M/PZ) or for 3H-pirenzepine binding in nine limbic thalamic nuclei and cingulate cortex. Specific OxO-M/PZ binding increased in the parvocellular division of the anterodorsal nucleus early in training when the animals were first exposed to pairing of the conditional and unconditional stimuli. Elevated binding in this nucleus was maintained throughout subsequent training. In the parvocellular division of the anteroventral nucleus (AVp), OxO-M/PZ binding progressively increased throughout training, reached a peak at the criterion stage of performance, and returned to control values during extinction sessions. Peak OxO-M/PZ binding in AVp was significantly elevated over that for cases yoked to criterion performance. In the magnocellular division of the anteroventral nucleus (AVm), OxO-M/PZ binding was elevated only during criterion performance of the task, and it was unaltered in any other limbic thalamic nuclei. Specific OxO-M/PZ binding was also elevated in most layers in rostral area 29c when subjects first performed a significant behavioral discrimination. Training-induced alterations in OxO-M/PZ binding in AVp and layer Ia of area 29c were similar and highly correlated

  17. Comparison of chemical binding to recombinant fathead minnow and human estrogen receptors alpha in whole cell and cell-free binding assays.

    Rider, Cynthia V; Hartig, Phillip C; Cardon, Mary C; Wilson, Vickie S

    2009-10-01

    Mammalian receptors and assay systems are generally used for in vitro screening of endocrine-disrupting chemicals with the assumption that minor differences in amino acid sequences among species do not translate into significant differences in receptor function. Objectives of the present study were to evaluate the performance of two different in vitro assay systems (a whole cell and a cell-free competitive binding assay) in assessing whether binding of chemicals differs significantly between full-length recombinant estrogen receptors from fathead minnows (fhERalpha) and those from humans (hERalpha). It was confirmed that 17beta-estradiol displays a reduction in binding to fhERalpha at an elevated temperature (37 degrees C), as has been reported with other piscine estrogen receptors. Several of the chemicals (17beta-estradiol, ethinylestradiol, alpha-zearalanol, fulvestrant, dibutyl phthalate, benzyl butyl phthalate, and cadmium chloride) displayed higher affinity for fhERalpha than for hERalpha in the whole cell assay, while only dibutyl phthalate had a higher affinity for fhERalpha than for hERalpha in the cell-free assay. Both assays were effective in identifying strong binders, weak binders, and nonbinders to the two receptors. However, the cell-free assay provided a less complicated and more efficient binding platform and is, therefore, recommended over the whole cell binding assay. In conclusion, no strong evidence showed species-specific binding among the chemicals tested. PMID:19453209

  18. Developmental profile of the aberrant dopamine D2 receptor response in striatal cholinergic interneurons in DYT1 dystonia.

    Giuseppe Sciamanna

    Full Text Available BACKGROUND: DYT1 dystonia, a severe form of genetically determined human dystonia, exhibits reduced penetrance among carriers and begins usually during adolescence. The reasons for such age dependence and variability remain unclear. METHODS AND RESULTS: We characterized the alterations in D2 dopamine receptor (D2R signalling in striatal cholinergic interneurons at different ages in mice overexpressing human mutant torsinA (hMT. An abnormal excitatory response to the D2R agonist quinpirole was recorded at postnatal day 14, consisting of a membrane depolarization coupled to an increase in spiking frequency, and persisted unchanged at 3 and 9 months in hMT mice, compared to mice expressing wild-type human torsinA and non-transgenic mice. This response was blocked by the D2R antagonist sulpiride and depended upon G-proteins, as it was prevented by intrapipette GDP-β-S. Patch-clamp recordings from dissociated interneurons revealed a significant increase in the Cav2.2-mediated current fraction at all ages examined. Consistently, chelation of intracellular calcium abolished the paradoxical response to quinpirole. Finally, no gross morphological changes were observed during development. CONCLUSIONS: These results suggest that an imbalanced striatal dopaminergic/cholinergic signaling occurs early in DYT1 dystonia and persists along development, representing a susceptibility factor for symptom generation.

  19. Why a diaminopyrrolic tripodal receptor binds mannosides in acetonitrile but not in water?

    Diogo Vila-Viçosa

    2014-07-01

    Full Text Available Intermolecular interactions involving carbohydrates and their natural receptors play important roles in several biological processes. The development of synthetic receptors is very useful to study these recognition processes. Recently, it was synthetized a diaminopyrrolic tripodal receptor that is selective for mannosides, which are obtained from mannose, a sugar with significant relevance in living systems. However, this receptor is significantly more active in acetonitrile than in water. In this work, we performed several molecular dynamics and constant-pH molecular dynamics simulations in acetonitrile and water to evaluate the conformational space of the receptor and to understand the molecular detail of the receptor–mannoside interaction. The protonation states sampled by the receptor show that the positive charges are always as distant as possible in order to avoid large intramolecular repulsions. Moreover, the conformational space of the receptor is very similar in water above pH 4.0 and in acetonitrile. From the simulations with the mannoside, we observe that the interactions are more specific in acetonitrile (mainly hydrogen bonds than in water (mainly hydrophobic. Our results suggest that the readiness of the receptor to bind mannoside is not significantly affected in water (above pH 4.0. Probably, the hydrogen bond network that is formed in acetonitrile (which is weaker in water is the main reason for the higher activity in this solvent. This work also presents a new implementation of the stochastic titration constant-pH molecular dynamics method to a synthetic receptor of sugars and attests its ability to describe the protonation/conformation coupling in these molecules.

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

    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.

  1. Measurement of biologically active interleukin-1 by a soluble receptor binding assay

    A soluble receptor binding assay has been developed for measuring human interleukin-1 alpha (IL-1 alpha), human IL-1 beta, and mouse IL-1 alpha. The assay is based on a competition between unlabeled IL-1 and 125I-labeled mouse recombinant IL-1 alpha for binding to soluble IL-1 receptor prepared from mouse EL-4 cells. The assay measures only biologically active IL-1 folded in its native conformation. The ratio of human IL-1 alpha to human IL-1 beta can be measured in the same sample by a pretreatment step which removes human IL-1 beta from samples prior to assay. This technique has been used to monitor the purification of recombinant IL-1, and may be utilized to specifically and accurately measure bioactive IL-1 in human serum and cell culture supernatants

  2. Differential binding of urokinase and peptide antagonists to the urokinase receptor

    Engelholm, L H; Behrendt, N

    2001-01-01

    though these sequences contain very few substitutions relative to the human uPAR, the receptor protein products differ markedly in terms of ligand selectivity. Thus, a well described competitive peptide antagonist directed against the human uPAR reacts with only one of the monkey receptors (chimpanzee u......PAR), in spite of the fact that uPAR from all of the four species cross-reacts with human uPA. Notably, uPAR from African green monkey, which is completely devoid of reactivity with the peptide, contains only three substitutions relative to chimpanzee uPAR in the molecular regions critical for binding....... These findings aid the elucidation of the structure/function relationship of uPAR and, unexpectedly, identify a structural distinction governing the binding of uPA and a very similar peptide antagonist....

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

    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.

  4. Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

    Bernstein, Lori R; Mackenzie, Amelia C L; Lee, Se-Jin; Chaffin, Charles L; Merchenthaler, István

    2016-03-01

    Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women. PMID:26713784

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

    1980-01-01

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

  6. Analysis of fluorescently labeled substance P analogs: binding, imaging and receptor activation

    Simmons Mark A

    2001-06-01

    Full Text Available Abstract Background Substance P (SP is a peptide neurotransmitter found in central and peripheral nerves. SP is involved in the control of smooth muscle, inflammation and nociception. The amino acid sequence of SP is Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. Five different forms of fluorescently labeled SP have recently been synthesized, in which Alexa 488, BODIPY Fl, fluorescein, Oregon Green 488 or tetramethylrhodamine has been covalently linked to SP at Lys3. Here, these novel analogs are characterized as to their ligand binding, receptor activation and fluorescence labeling properties. Results Competition binding studies, using radiolabeled [125I] SP, revealed that all of the labeled forms of SP, except for Alexa 488-SP, effectively competed with radiolabeled SP for binding at the rat SP receptor. With the exception of Alexa 488-SP, all of the SP analogs produced Ca++ elevations and fluorescence labeling of the SP receptor expressed in Chinese hamster ovary cells. In SP-responsive neurons, BODIPY Fl-SP and Oregon Green 488-SP were as effective as unlabeled SP in producing a reduction of the M-type K+ current. Fluorescein-SP produced variable results, while tetramethylrhodamine-SP was less potent and Alexa 488-SP was less effective on intact neurons. Conclusions The above results show that fluorescent labeling of SP altered the biological activity and the binding properties of the parent peptide. Oregon Green 488 and BODIPY FL-SP are the most useful fluorophores for labeling SP without affecting its biological activity. Given these results, these probes can now be utilized in further investigations of the mechanisms of SPR function, including receptor localization, internalization and recycling.

  7. Blocking and Binding Folate Receptor Alpha Autoantibodies Identify Novel Autism Spectrum Disorder Subgroups

    Frye, Richard E; Delhey, Leanna; Slattery, John; Tippett, Marie; Wynne, Rebecca; Rose, Shannon; Kahler, Stephen G.; Bennuri, Sirish C.; Melnyk, Stepan; Sequeira, Jeffrey M.; Quadros, Edward

    2016-01-01

    Folate receptor α (FRα) autoantibodies (FRAAs) are prevalent in autism spectrum disorder (ASD). They disrupt the transportation of folate across the blood-brain barrier by binding to the FRα. Children with ASD and FRAAs have been reported to respond well to treatment with a form of folate known as folinic acid, suggesting that they may be an important ASD subgroup to identify and treat. There has been no investigation of whether they manifest unique behavioral and physiological characteristic...

  8. Kinetics of leptin binding to the Q223R leptin receptor.

    Hans Verkerke

    Full Text Available Studies in human populations and mouse models of disease have linked the common leptin receptor Q223R mutation to obesity, multiple forms of cancer, adverse drug reactions, and susceptibility to enteric and respiratory infections. Contradictory results cast doubt on the phenotypic consequences of this variant. We set out to determine whether the Q223R substitution affects leptin binding kinetics using surface plasmon resonance (SPR, a technique that allows sensitive real-time monitoring of protein-protein interactions. We measured the binding and dissociation rate constants for leptin to the extracellular domain of WT and Q223R murine leptin receptors expressed as Fc-fusion proteins and found that the mutant receptor does not significantly differ in kinetics of leptin binding from the WT leptin receptor. (WT: ka 1.76×106±0.193×106 M-1 s-1, kd 1.21×10-4±0.707×10-4 s-1, KD 6.47×10-11±3.30×10-11 M; Q223R: ka 1.75×106±0.0245×106 M-1 s-1, kd 1.47×10-4±0.0505×10-4 s-1, KD 8.43×10-11±0.407×10-11 M. Our results support earlier findings that differences in affinity and kinetics of leptin binding are unlikely to explain mechanistically the phenotypes that have been linked to this common genetic variant. Future studies will seek to elucidate the mechanism by which this mutation influences susceptibility to metabolic, infectious, and malignant pathologies.

  9. Neonicotinoid Binding, Toxicity and Expression of Nicotinic Acetylcholine Receptor Subunits in the Aphid Acyrthosiphon pisum

    Taillebois, Emiliane; Beloula, Abdelhamid; Quinchard, Sophie; Jaubert-Possamai, Stéphanie; Daguin, Antoine; Servent, Denis; Tagu, Denis; Thany, Steeve H.; Tricoire-Leignel, Helene

    2014-01-01

    Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and ...

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

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

    2011-01-01

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

  11. Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering.

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Studer, Romain A; Alvarez, Susana; de Lera, Angel R; Kuraku, Shigehiro; Bourguet, William; Schubert, Michael; Laudet, Vincent

    2016-03-01

    Whole genome duplications (WGDs) have been classically associated with the origin of evolutionary novelties and the so-called duplication-degeneration-complementation model describes the possible fates of genes after duplication. However, how sequence divergence effectively allows functional changes between gene duplicates is still unclear. In the vertebrate lineage, two rounds of WGDs took place, giving rise to paralogous gene copies observed for many gene families. For the retinoic acid receptors (RARs), for example, which are members of the nuclear hormone receptor (NR) superfamily, a unique ancestral gene has been duplicated resulting in three vertebrate paralogues: RARα, RARβ and RARγ. It has previously been shown that this single ancestral RAR was neofunctionalized to give rise to a larger substrate specificity range in the RARs of extant jawed vertebrates (also called gnathostomes). To understand RAR diversification, the members of the cyclostomes (lamprey and hagfish), jawless vertebrates representing the extant sister group of gnathostomes, provide an intermediate situation and thus allow the characterization of the evolutionary steps that shaped RAR ligand-binding properties following the WGDs. In this study, we assessed the ligand-binding specificity of cyclostome RARs and found that their ligand-binding pockets resemble those of gnathostome RARα and RARβ. In contrast, none of the cyclostome receptors studied showed any RARγ-like specificity. Together, our results suggest that cyclostome RARs cover only a portion of the specificity repertoire of the ancestral gnathostome RARs and indicate that the establishment of ligand-binding specificity was a stepwise event. This iterative process thus provides a rare example for the diversification of receptor-ligand interactions of NRs following WGDs. PMID:27069642

  12. Glucose regulates fatty acid binding protein interaction with lipids and peroxisome proliferator-activated receptor α

    Hostetler, Heather A.; Balanarasimha, Madhumitha; Huang, Huan; Kelzer, Matthew S.; Kaliappan, Alagammai; Kier, Ann B.; Schroeder, Friedhelm

    2010-01-01

    Although the pathophysiology of diabetes is characterized by elevated levels of glucose and long-chain fatty acids (LCFA), nuclear mechanisms linking glucose and LCFA metabolism are poorly understood. As the liver fatty acid binding protein (L-FABP) shuttles LCFA to the nucleus, where L-FABP directly interacts with peroxisome proliferator-activated receptor-α (PPARα), the effect of glucose on these processes was examined. In vitro studies showed that L-FABP strongly bound glucose and glucose-...

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

    Özge Gündüz Çınar; Hasan Kırmızıbekmez; Galip Akaydın; Erdem Yesilada

    2011-01-01

    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 recept...

  14. Ciguatera risk assessment in two toxic sites of French Polynesia using the receptor-binding assay

    Darius, H.T.; Ponton, Dominique; De Revel, T.; Cruchet, P.; A. Ung; Fouc, M. T.; Chinain, M. (Mireille)

    2007-01-01

    Ciguatera Fish Poisoning (CFP) is a tropical syndrome well known in remote archipelagos where the population is still dependent on fish resources. In order to assess the ciguatera risk in two islands of French Polynesia, Tubuai (Australes) and Nuku Hiva (Marquesas), a study was carried out on both Gambierdiscus populations as well as on various fish species using the receptor-binding assay (RBA) to detect and quantify ciguatoxins. Relationship between RBA data and size or weight of fish was e...

  15. Pathway Analysis Revealed Potential Diverse Health Impacts of Flavonoids that Bind Estrogen Receptors

    Ye, Hao; NG, HUI WEN; Sakkiah, Sugunadevi; Ge, Weigong; Perkins, Roger; Tong, Weida; Hong, Huixiao

    2016-01-01

    Flavonoids are frequently used as dietary supplements in the absence of research evidence regarding health benefits or toxicity. Furthermore, ingested doses could far exceed those received from diet in the course of normal living. Some flavonoids exhibit binding to estrogen receptors (ERs) with consequential vigilance by regulatory authorities at the U.S. EPA and FDA. Regulatory authorities must consider both beneficial claims and potential adverse effects, warranting the increases in researc...

  16. Structure-based rational design of a Toll-like receptor 4 (TLR4 decoy receptor with high binding affinity for a target protein.

    Jieun Han

    Full Text Available Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4 decoy receptor composed of leucine-rich repeat (LRR modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2. Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (K(D one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities.

  17. Nuclear triiodothyronine receptor binding characteristics and occupancy in obese (ob/ob) mice

    Hillgartner, F.B.; Romsos, D.R.

    1987-03-01

    Obese (ob/ob) mice exhibit reduced adaptive thermogenesis associated with an impairment of thyroid hormone action. The mechanism underlying the latter defect was investigated by comparing the binding characteristics and occupancy of solubilized nuclear 3,5,3'-triiodothyronine (T/sub 3/) receptors from livers of lean and obese mice. T/sub 3/ concentration was measured by radioimmunoassay. Scatchard analysis showed minimal differences in B/sub max/ and K/sub d/ between phenotypes at both 4 and 8-10 wk of age, indicating that reduced hepatic thyroid hormone expression in obese mice is not caused by alterations in nuclear receptor concentration or affinity. In contrast, nuclear T/sub 3/ receptor occupancy (endogenous T/sub 3/ associated with the specific receptor divided by B/sub max/) was 14 and 23% lower in 4- and 8- to 10-wk old obese mice, respectively. Together with reported changes in hepatic thyroid hormone-sensitive enzymes, these data are consistent with a diminished nuclear T/sub 3/ signal initiating thyroid hormone action in obese mice. Decreased nuclear T/sub 3/ receptor occupancy may be secondary to a low transport of plasma T/sub 3/ to the nuclear pool. In conclusion, impaired hepatic thyroid hormone action in obese mice is mediated in part at least by a reduction in nuclear T/sub 3/ receptor occupancy.

  18. Nuclear triiodothyronine receptor binding characteristics and occupancy in obese (ob/ob) mice

    Obese (ob/ob) mice exhibit reduced adaptive thermogenesis associated with an impairment of thyroid hormone action. The mechanism underlying the latter defect was investigated by comparing the binding characteristics and occupancy of solubilized nuclear 3,5,3'-triiodothyronine (T3) receptors from livers of lean and obese mice. T3 concentration was measured by radioimmunoassay. Scatchard analysis showed minimal differences in B/sub max/ and K/sub d/ between phenotypes at both 4 and 8-10 wk of age, indicating that reduced hepatic thyroid hormone expression in obese mice is not caused by alterations in nuclear receptor concentration or affinity. In contrast, nuclear T3 receptor occupancy (endogenous T3 associated with the specific receptor divided by B/sub max/) was 14 and 23% lower in 4- and 8- to 10-wk old obese mice, respectively. Together with reported changes in hepatic thyroid hormone-sensitive enzymes, these data are consistent with a diminished nuclear T3 signal initiating thyroid hormone action in obese mice. Decreased nuclear T3 receptor occupancy may be secondary to a low transport of plasma T3 to the nuclear pool. In conclusion, impaired hepatic thyroid hormone action in obese mice is mediated in part at least by a reduction in nuclear T3 receptor occupancy

  19. Competitive receptor binding radioassay for β-1 and β-2 adrenergic agents

    A rapid and sensitive competitive receptor bonding assay for β-1 and β-2 adrenergic binding for adrenergic agents has been developed. The steps that are critical for the success of the assay are given in detail so that the assay can be set up in any routine laboratory with relative ease. The rationale behind the use of specific reagents is discussed. The assay requires microgram quantities of test compound, a radiolabeled specific β adrenergic antagonist [3H]dihydroalprenolol (DHA), and turkey erythrocyte β-1 and rat erythrocyte β-2 receptor membranes. Serial dilutions of sample are incubated with appropriate receptor membranes and DHA for 1 hr at room temperature. After equilibrium is attained, the bound radioligand is separated by rapid filtration under vacuum through Whatman GF/B filters. The amount of bound DHA trapped on the filter is inversely proportional to the degree of β-1 and β-2 adrenergic binding of the sample. Separation of bound from free radioligand by filtration permits rapid determination of a large number of samples. This assay quantitates and differentiates β-1 and β-2 adrenergic binding of synthetic adrenergic agents

  20. Active regions' setting of the extracellular ligand-binding domain of human interleukin-6 receptor

    2000-01-01

    The reliable three dimensional (3-D) structure of the extracellular ligand-binding domain (V106-P322) of human interleukin-6 receptor (hIL-6R) has been constructed by means of computer-guided homology modeling techniques using the crystal structure of the extracellular ligand-binding region (K52-L251) of human growth hormone receptor (hGHR) as templet. The space location of some key residues which influence the combination ability between the receptor and the ligand has been observed and the effects of point mutagenesis of the four conservative cysteine residues on the space conformation are analyzed. The results show that the space conformation of the side-chain carboxyl of E305 plays a key role in the ligand-binding ability. Furthermore, the space conformation of the side-chain carboxyl of E305 is very important for the electrostatic potential complementarity between hIL-6R and hIL-6 according to the docking method.

  1. Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

    The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer

  2. Chronic brief restraint decreases in vivo binding of benzodiazepine receptor ligand to mouse brain.

    Mosaddeghi, M; Burke, T F; Moerschbaecher, J M

    1993-01-01

    This study examines the effects of chronic brief restraint on in vivo benzodiazepine (BZD) receptor binding in mouse brain. Three groups of mice were used. Mice in group 1 were neither restrained nor injected (ACUTE control). Mice in group 2 were restrained for 5-6 s by grabbing the back skin and holding the subject upside-down at a 45 degrees angle as if to be injected (CHRONIC SHAM control) for 7 d. Mice in group 3 (CHRONIC SALINE) received daily single intraperitoneal (ip) injections of saline (5 mL/kg) for 7 d. On d 8 BZD receptors were labeled in vivo by administration of 3 microCi [3H]flumazenil (ip). The levels of ligand bound in vivo to cerebral cortex (CX), cerebellum (CB), brain stem (BS), striatum (ST), hippocampus (HP), and hypothalamus (HY) were determined. Results indicated that the level of binding was significantly (p stress produces a decrease in BZD receptor binding sites. PMID:8385464

  3. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    Marlet Martinez-Archundia

    2012-01-01

    Full Text Available The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS. Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

  4. Low density lipoprotein receptor-binding activity in human tissues: Quantitative importance of hepatic receptors and evidence for regulation of their expression in vivo

    Rudling, M.J. (Karolinska Instituet at Huddinge University Hospital (Sweden) Karolinska Institutet, Stockholm (Sweden)); Reihner, E.; Einarsson, K.; Ewerth, S.; Angelin, B. (Karolinska Institutet at Huddinge University Hospital (Sweden))

    1990-05-01

    The heparin-sensitive binding of {sup 125}I-labeled low-density lipoprotein (LDL) to homogenates from 18 different normal human tissues and some solid tumors was determined. The binding to adrenal and liver homogenates fulfilled criteria established for the binding of LDL to its receptor--namely, (i) saturability, (ii) sensitivity to proteolytic destruction, (iii) inhibition by EDTA, and (iv) heat sensitivity. When the binding of {sup 125}I-labeled LDL was assayed at a constant concentration, the adrenal gland and the ovary had the highest binding of normal tissues. The highest binding per g of tissue overall was obtained in homogenates of a gastric carcinoma and a parotid adenoma. When the weights of the parenchymatous organs were considered, the major amount of LDL receptors was contained in the liver. To study the possible regulation of hepatic LDL-receptor expression, 11 patients were pretreated with cholestyramine. Increased binding activity was obtained in homogenates from liver biopsies from the cholestyramine-treated patients as compared with 12 untreated controls. It is concluded that the liver is the most important organ for LDL catabolism in humans and that the receptor activity in this organ can be regulated upon pharmacologic intervention. Further studies are needed to confirm the possibility that certain solid tumors can exhibit high numbers of LDL receptors.

  5. Low density lipoprotein receptor-binding activity in human tissues: Quantitative importance of hepatic receptors and evidence for regulation of their expression in vivo

    The heparin-sensitive binding of 125I-labeled low-density lipoprotein (LDL) to homogenates from 18 different normal human tissues and some solid tumors was determined. The binding to adrenal and liver homogenates fulfilled criteria established for the binding of LDL to its receptor--namely, (i) saturability, (ii) sensitivity to proteolytic destruction, (iii) inhibition by EDTA, and (iv) heat sensitivity. When the binding of 125I-labeled LDL was assayed at a constant concentration, the adrenal gland and the ovary had the highest binding of normal tissues. The highest binding per g of tissue overall was obtained in homogenates of a gastric carcinoma and a parotid adenoma. When the weights of the parenchymatous organs were considered, the major amount of LDL receptors was contained in the liver. To study the possible regulation of hepatic LDL-receptor expression, 11 patients were pretreated with cholestyramine. Increased binding activity was obtained in homogenates from liver biopsies from the cholestyramine-treated patients as compared with 12 untreated controls. It is concluded that the liver is the most important organ for LDL catabolism in humans and that the receptor activity in this organ can be regulated upon pharmacologic intervention. Further studies are needed to confirm the possibility that certain solid tumors can exhibit high numbers of LDL receptors

  6. Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding.

    Schwarz, Rico; Tänzler, Dirk; Ihling, Christian H; Sinz, Andrea

    2016-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors. PMID:26992147

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

    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

  8. Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region

    Madsen, Mette; Møller, Holger J; Nielsen, Marianne Jensby;

    2004-01-01

    CD163 is the macrophage receptor for endocytosis of haptoglobin.hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteine rich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163...

  9. Sexually dimorphic development and binding characteristics of NMDA receptors in the brain of the platyfish

    Flynn, K. M.; Schreibman, M. P.; Yablonsky-Alter, E.; Banerjee, S. P.

    1999-01-01

    This study investigated age- and gender-specific variations in properties of the glutamate N-methyl-d-aspartate receptor (NMDAR) in a freshwater teleost, the platyfish (Xiphophorus maculatus). Prior localization of the immunoreactive (ir)-R1 subunit of the NMDAR protein (R1) in cells of the nucleus olfactoretinalis (NOR), a primary gonadotropin-releasing hormone (GnRH)-containing brain nucleus in the platyfish, suggests that NMDAR, as in mammals, is involved in modulation of the platyfish brain-pituitary-gonad (BPG) axis. The current study shows that the number of cells in the NOR displaying ir-R1 is significantly increased in pubescent and mature female platyfish when compared to immature and senescent animals. In males, there is no significant change in ir-R1 expression in the NOR at any time in their lifespan. The affinity of the noncompetitive antagonist ((3)H)MK-801 for the NMDAR is significantly increased in pubescent females while maximum binding of ((3)H)MK-801 to the receptor reaches a significant maximum in mature females. In males, both MK-801 affinity and maximum binding remain unchanged throughout development. This is the first report of gender differences in the association of NMDA receptors with neuroendocrine brain areas during development. It is also the first report to suggest NMDA receptor involvement in the development of the BPG axis in a nonmammalian vertebrate. Copyright 1999 Academic Press.

  10. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  11. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    The mechanism by which delta9 tetrahydrocannabinol (delta9THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5'-Trimethylammonium-delta8THC (TMA) is a positively charged analog of delta-8THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of [3H]-5'-trimethylammonium-delta-8THC ([3H]TMA) to rat neuronal membranes. [3H]TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of [3H]TMA binding activity of approximately 60,000 daltons apparent molecular weight

  12. The Hinge Region of Human Thyroid-Stimulating Hormone (TSH) Receptor Operates as a Tunable Switch between Hormone Binding and Receptor Activation

    Majumdar, Ritankar; Dighe, Rajan R.

    2012-01-01

    The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone ...

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

    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. Binding of [3H]ethyl-β-carboline-3-carboxylate to brain benzodiazepine receptors

    It is reported that in contrast to the changes in affinity of [3H]benzodiazepines elicited by halide ions, barbiturates, and pyrazolopyridines, the apparent affinity of β-[3H]CCE (ethyl-β-carboline-3-carboxylate) is unaffected by these agents. Furthermore, Scatchard analysis of β-[3H]CCE binding to cerebral cortical and cerebellar membranes revealed a significantly greater number of binding sites than was observed with either [3H]diazepam or [3H]flunitazepam, suggesting that at low concentrations benzodiazepines selectively label a subpopulation of the receptors labelled with β-[3H]CCE. Alternatively, β-[3H]CCE may bind to sites that are distinct from those labelled with [3H]-benzodiazepines. (Auth.)

  15. Cerebral 5-HT2A receptor binding is increased in patients with Tourette's syndrome

    Haugbøl, Steven; Pinborg, Lars H.; Regeur, Lisbeth; Hansen, Elsebet S.; Bolwig, Tom G.; Nielsen, Finn Årup; Svarer, Claus; Skovgaard, Lene T.; Knudsen, Gitte Moos

    2007-01-01

    Experimental and clinical data have suggested that abnormalities in the serotonergic neurotransmissions in frontal-subcortical circuits are involved in Tourette's syndrome. To test the hypothesis that the brain's 5-HT2A receptor binding is increased in patients with Tourette's syndrome, PET imaging...... was performed. Twenty adults with Tourette's syndrome and 20 healthy control subjects were investigated with PET-[18F]altanserin using a bolus-infusion protocol. Regions of interest were delineated automatically on co-registered MRI images, and partial volume-corrected binding parameters were...... extracted from the PET images. Comparison between control subjects and Tourette's syndrome patients showed increased specific [18F]altanserin binding, not only in the a-priori selected brain regions hypothesized to be involved in Tourette's syndrome, but also post-hoc analysis showed a global up...

  16. Locomotor activity and catecholamine receptor binding in adult normotensive and spontaneously hypertensive rats

    The binding of 3H-WB 4101, an α1-adrenoceptor antagonist, the membranes of the cerebral cortex, the hypothalamus, and the lower brainstem was examined in adult spontaneously hypertensive (SH) rats and in normotensive Wistar Kyoto (WK) controls. The specific binding of 3H-WB 4101 (0.33 nM) was significantly higher in homogenates from the cerebral cortex of SH rats as compared to WK rats. No differences were detected between SH and WK rats in the specific binding of 3H-spiroperidol (0.25 nM), a dopamine receptor antagonist, to membranes from the corpus striatum and the limbic forebrain. The locomotor activity was significantly higher in SH rats as compared to WK controls, in all probability due to a lack of habituation to environmental change. It is suggested that the high reactivity of SH rats is related to a disfunction in the noradrenergic neurons in the central nervous system. (author)

  17. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M. PMID:23325100

  18. Ligand Binding Affinities of Arctigenin and Its Demethylated Metabolites to Estrogen Receptor Alpha

    Masao Hattori

    2013-01-01

    Full Text Available Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (−-arctigenin, the aglycone of arctiin, was demethylated to (−-dihydroxyenterolactone (DHENL by Eubacterium (E. sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (−-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (−-arctigenin using a ligand binding screen assay method. The IC50 value of (2R,3R-2-(4-hydroxy-3-methoxybenzyl-3-(3,4-dihydroxybenzyl-butyrolactone was 7.9 × 10−4 M.

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

    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. PMID:18840687

  20. Increased dopamine D1 receptor binding in the human mesocortical system following central cholinergic activation

    Full text: The interaction between the cholinergic and dopaminergic system has been implicated in many pathological processes including, Alzheimer's disease, schizophrenia and drug addiction. Little is known about the control of dopamine (DA) release following central cholinergic activation in humans, but experimental studies suggest that endogenously released Acetylcholine (ACh) achieved by the administration of cholinesterase inhibitors, can increase dopamine efflux in different regions of the brain. This leads to the activation of different types of post-synaptic dopaminergic receptors which belong to the family of G-protein coupled receptors (GPCRs). A common paradigm of the GPCRs desensitization is that agonist-induced receptor signaling is rapidly attenuated by receptor internalisation. Several experiments have shown that the activation of Dl receptors in acute conditions leads, within minutes, to translocation of the receptor from the surface of the neurons to the endosomal compartment in the cytoplasm and increased receptor turnover. To assess changes in Dl receptor density following an intravenous infusion of the selective cholinesterase inhibitor physostigmine salicylate (PHY), we studied eleven normal subjects (10 male and 1 female, mean age 36.1 and 61617; 9.9) using [11C]-SCH23390 and PET The binding potential (BP) for SCH23390 was significantly (p0.05). There was no statistically significant difference between baseline and physostigmine Kl ratio (p>0.05) suggesting that BP changes observed were not secondary to regional blood flow changes or to an order effect of the scans. Copyright (2002) The Australian and New Zealand Society of Nuclear Medicine Inc

  1. Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens

    Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17β to the four rainbow trout ER isoforms with that of three known environmental estrogens 17α-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ERα subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17β, bisphenol A binds less strongly to all four receptors, 17α-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the α subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.

  2. Substituted benzamides as ligands for visualization of dopamine receptor binding in the human brain by positron emission tomography

    Two substituted benzamides, FLB 524 and raclopride, were labeled with 11C and examined for their possible use as ligands for positron emission tomography (PET) scan studies on dopamine-2 (D-2) receptors in the brains of monkeys and healthy human subjects. Both ligands allowed the in vivo visualization of D-2 receptor binding in the corpus striatum caudate nucleus/putamen complex in PET-scan images. [11C]Raclopride showed a high ratio of specific striatal to nonspecific cerebellar binding, and the kinetics of binding of this ligand made it optimal for PET studies. The in vivo binding of [11C]raclopride in the striatum of cynomolgus monkeys was markedly reduced by displacement with haloperidol. In healthy human subjects, [11C]raclopride binding in the caudate nucleus/putamen was 4- to 5-fold greater than nonspecific binding in the cerebellum. In comparison with previously available ligands for PET-scan studies on central dopamine receptors in man, [11C]raclopride appears to be advantageous with regard to (i) specificity of binding to D-2 receptors, (ii) the high ratio between binding in dopamine-rich (caudate, putamen) and dopamine-poor (cerebellum) human brain regions, and (iii) rapid association and reversibility of specific binding

  3. Improved binding affinity and interesting selectivities of aminopyrimidine-bearing carbohydrate receptors in comparison with their aminopyridine analogues.

    Lippe, Jan; Seichter, Wilhelm; Mazik, Monika

    2015-12-28

    Due to the problems with the exact prediction of the binding properties of an artificial carbohydrate receptor, the identification of characteristic structural features, having the ability to influence the binding properties in a predictable way, is of high importance. The purpose of our investigation was to examine whether the previously observed higher affinity of 2-aminopyrimidine-bearing carbohydrate receptors in comparison with aminopyridine substituted analogues represents a general tendency of aminopyrimidine-bearing compounds. Systematic binding studies on new compounds consisting of 2-aminopyrimidine groups confirmed such a tendency and allowed the identification of interesting structure-activity relationships. Receptors having different symmetries showed systematic preferences for specific glycosides, which are remarkable for such simple receptor systems. Particularly suitable receptor architectures for the recognition of selected glycosides were identified and represent a valuable base for further developments in this field. PMID:26467387

  4. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    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 extracellular 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)

  5. Relationship of Structure and Function of DNA-Binding Domain in Vitamin D Receptor

    Lin-Yan Wan

    2015-07-01

    Full Text Available While the structure of the DNA-binding domain (DBD of the vitamin D receptor (VDR has been determined in great detail, the roles of its domains and how to bind the motif of its target genes are still under debate. The VDR DBD consists of two zinc finger modules and a C-terminal extension (CTE, at the end of the C-terminal of each structure presenting α-helix. For the first zinc finger structure, N37 and S-box take part in forming a dimer with 9-cis retinoid X receptor (RXR, while V26, R50, P-box and S-box participate in binding with VDR response elements (VDRE. For the second zinc finger structure, P61, F62 and H75 are essential in the structure of the VDR homodimer with the residues N37, E92 and F93 of the downstream of partner VDR, which form the inter-DBD interface. T-box of the CTE, especially the F93 and I94, plays a critical role in heterodimerization and heterodimers–VDRE binding. Six essential residues (R102, K103, M106, I107, K109, and R110 of the CTE α-helix of VDR construct one interaction face, which packs against the DBD core of the adjacent symmetry mate. In 1,25(OH2D3-activated signaling, the VDR-RXR heterodimer may bind to DR3-type VDRE and ER9-type VDREs of its target gene directly resulting in transactivation and also bind to DR3-liked nVDRE of its target gene directly resulting in transrepression. Except for this, 1α,25(OH2D3 ligand VDR-RXR may bind to 1αnVDRE indirectly through VDIR, resulting in transrepression of the target gene. Upon binding of 1α,25(OH2D3, VDR can transactivate and transrepress its target genes depending on the DNA motif that DBD binds.

  6. DIFFERENTIAL BINDING OF HUMAN INTERLEUKIN-1 (IL-1) RECEPTOR ANTAGONIST TO NATURAL AND RECOMBINANT SOLUBLE AND CELLULAR IL-1 TYPE-I RECEPTORS

    Svenson, Morten; Nedergaard, Susanne; Heegaard, Peter M. H.;

    1995-01-01

    A recently described factor, interleukin-1 receptor antagonist binding factor (IL-1raBF), in serum of normal individuals is immunologically related to the interleukin-1 receptor type I (IL-1RI). It is presumably a soluble form of the receptor that binds exclusively to interleukin-1 receptor...... antagonist (IL-1ra). Recombinant soluble human IL-1RI expressed in COS cells (sIL-1RI) consists of the extracellular part of the receptor and binds all three known IL-1 species but preferentially to IL-1ra. We further characterized the sizes and binding of IL-1raBF and sIL-1RI to IL-1ra by polyacrylamide gel...... electrophoresis in the presence of sodium dodecylsulfate, ligand binding interference analyses, N-glycosidase treatment, concanavalin A affinity chromatography, and with the use of monoclonal antibodies (mAb) to human recombinant IL-1ra. We also evaluated the binding of IL-1ra to cellular IL-1RI on MRC5...

  7. Receptor-binding region in human choriogonadotropin/lutropin β subunit

    Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. The authors prepared a series of peptides representing the intercysteine loop sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) β subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCGβ-(38-57) and hLHβ-(38-57) peptides inhibited binding of 125I-labeled hCG half-maximally at 1.51 x 10-4 and 2.03 x 10-5 M, respectively, while other peptide hormones and fragments from elsewhere in the β subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 x 10-5 M (hCG) and 2.18 x 10-5 M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCGβ-(38-57) peptide, native hCG and β subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. These results indicate that the 38-57 region of β subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones

  8. (+)-Cannabidiol analogues which bind cannabinoid receptors but exert peripheral activity only.

    Fride, Ester; Feigin, Cfir; Ponde, Datta E; Breuer, Aviva; Hanus, Lumír; Arshavsky, Nina; Mechoulam, Raphael

    2004-12-15

    Delta9-Tetrahydrocannabinol (Delta9-THC) and (-)-cannabidiol are major constituents of the Cannabis sativa plant with different pharmacological profiles: (-)-Delta9-tetrahydrocannabinol, but not (-)-cannabidiol, activates cannabinoid CB1 and CB2 receptors and induces psychoactive and peripheral effects. We have tested a series of (+)-cannabidiol derivatives, namely, (+)-cannabidiol-DMH (DMH-1,1-dimethylheptyl-), (+)-7-OH-cannabidiol-DMH, (+)-7-OH- cannabidiol, (+)-7-COOH- cannabidiol and (+)-7-COOH-cannabidiol-DMH, for central and peripheral (intestinal, antiinflammatory and peripheral pain) effects in mice. Although all (+)-cannabidiols bind to cannabinoid CB1 and CB2 receptors, only (+)-7-OH-cannabidiol-DMH was centrally active, while all (+)-cannabidiol analogues completely arrested defecation. The effects of (+)-cannabidiol-DMH and (+)-7-OH-cannabidiol-DMH were partially antagonized by the cannabinoid CB1 receptor antagonist N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716), but not by the cannabinoid CB2 receptor antagonist N-[-(1S)-endo-1,3,3-trimethil bicyclo [2.2.1] heptan-2-yl-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), and had no effect on CB1(-/-) receptor knockout mice. (+)-Cannabidiol-DMH inhibited the peripheral pain response and arachidonic-acid-induced inflammation of the ear. We conclude that centrally inactive (+)-cannabidiol analogues should be further developed as antidiarrheal, antiinflammatory and analgesic drugs for gastrointestinal and other peripheral conditions. PMID:15588739

  9. Ritonavir binds to and downregulates estrogen receptors: Molecular mechanism of promoting early atherosclerosis

    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.

  10. Thermodynamics of the interaction between oxytocin and its myometrial receptor in sheep: a stepwise binding mechanism.

    Pliska, Vladimir; Folkers, Gerd; Spiwok, Vojtěch

    2014-09-01

    Entropy (ΔS), enthalpy (ΔH) and heat capacity (ΔCp) changes attending the oxytocin interaction with its two binding sites on myometrial cell membranes in sheep were derived from the temperature dependence of Kd values. The high affinity oxytocin site (Kd on the order of 10(-9)mol l(-1), 25 °C), ascribed to the oxytocin receptor (OXTR), is entropy-driven in the temperature range 0-37 °C. Enthalpy component prevails as a driving force in the binding to the low affinity site (Kd ≈ 10(-7)) within the higher temperature range. ΔCp values in both cases do not differ significantly from zero but become highly relevant in the presence of a GTP analog (10(-4)M GTP-γS). Under these conditions, ΔCp in the low site interaction becomes negative and ΔS is shifted toward negative values (enthalpy drift); ΔCp of the high affinity site rises to a high positive value and the interaction is even more strongly entropy driven. Atosiban, a competitive antagonist of oxytocin at OXTR displays a single significant binding site on myometrial cells (Kd about 10(-7)mol l(-1)). Thermodynamic profiles of atosiban and the low affinity oxytocin site show conspicuous similarities, indicating that the inhibitor is bound to the low affinity site, and not, with a lower affinity, to the putative receptor protein. It is suggested that the interaction of oxytocin with its responding system on myometrial membranes follows in two distinct steps that are likely to be associated with several independent binding domains in the GPCR receptor. PMID:25010721

  11. Ritonavir binds to and downregulates estrogen receptors: Molecular mechanism of promoting early atherosclerosis

    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

  12. Enhanced binding of morphine and nalorphine to opioid delta receptor by glucuronate and sulfate conjugations at the 6-position

    Oguri, K.; Yamada-Mori, I.; Shigezane, J.; Hirano, T.; Yoshimura, H.

    1987-09-21

    Effect of the modification of morphine and nalorphine by glucuronate and sulfate conjugations at the 3- and 6-positions on the binding to opioid receptors was examined in a particulate fraction of rat brain. Competing potencies of both drugs against (/sup 3/H)morphine and (/sup 3/H)leucine enkephalin bindings were extremely decreased by either glucuronate or sulfate conjugation at the 3-position. On the other hand, the potencies of morphine and nalorphine against (/sup 3/H)leucine enkephalin binding were considerably enhanced by the conjugations at the 6-position, whereas the potencies against (/sup 3/H)morphine binding were decreased. These altered interactions of the conjugates at the 6-position with the two ligands were attributed to their enhanced binding to delta-receptor and reduced binding to /sup +/-receptor by Hill plot and modified Scatchard analysis. Resulted comparable and simultaneous interactions with ..mu..- and delta- receptors were assumed to be a cause of the enhanced ..mu..-receptor-directed analgesia of morphine and elevated same receptor-directed antagonistic effect of nalorphine, which have been found previously in the authors laboratory. 24 references, 2 figures, 1 table.

  13. Enhanced binding of morphine and nalorphine to opioid delta receptor by glucuronate and sulfate conjugations at the 6-position

    Effect of the modification of morphine and nalorphine by glucuronate and sulfate conjugations at the 3- and 6-positions on the binding to opioid receptors was examined in a particulate fraction of rat brain. Competing potencies of both drugs against [3H]morphine and [3H]leucine enkephalin bindings were extremely decreased by either glucuronate or sulfate conjugation at the 3-position. On the other hand, the potencies of morphine and nalorphine against [3H]leucine enkephalin binding were considerably enhanced by the conjugations at the 6-position, whereas the potencies against [3H]morphine binding were decreased. These altered interactions of the conjugates at the 6-position with the two ligands were attributed to their enhanced binding to δ-receptor and reduced binding to +-receptor by Hill plot and modified Scatchard analysis. Resulted comparable and simultaneous interactions with μ- and δ- receptors were assumed to be a cause of the enhanced μ-receptor-directed analgesia of morphine and elevated same receptor-directed antagonistic effect of nalorphine, which have been found previously in the authors laboratory. 24 references, 2 figures, 1 table

  14. Platelet alpha 2-adrenergic receptors in major depressive disorder. Binding of tritiated clonidine before and after tricyclic antidepressant drug treatment

    The specific binding of tritiated (3H)-clonidine, an alpha 2-adrenergic receptor agonist, to platelet membranes was measured in normal subjects and in patients with major depressive disorder. The number of platelet alpha 2-adrenergic receptors from the depressed group was significantly higher than that found in platelets obtained from the control population. Treatment with tricyclic antidepressant drugs led to significant decreases in the number of platelet alpha 2-adrenergic receptors. These results support the hypothesis that the depressive syndrome is related to an alpha 2-adrenergic receptor supersensitivity and that the clinical effectiveness of tricyclic antidepressant drugs is associated with a decrease in the number of these receptors

  15. High-Mobility Group Chromatin Proteins 1 and 2 Functionally Interact with Steroid Hormone Receptors To Enhance Their DNA Binding In Vitro and Transcriptional Activity in Mammalian Cells

    Boonyaratanakornkit, Viroj; Melvin, Vida; Prendergast, Paul; Altmann, Magda; Ronfani, Lorenza; Marco E. Bianchi; Taraseviciene, Laima; Nordeen, Steven K.; Allegretto, Elizabeth A.; Edwards, Dean P.

    1998-01-01

    We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by se...

  16. Structural Analysis of the Ligand-Binding Domain of the Aspartate Receptor Tar from Escherichia coli.

    Mise, Takeshi

    2016-07-01

    The Escherichia coli cell-surface aspartate receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). These signals are transmitted from the extracellular region of Tar to the cytoplasmic region via the transmembrane domain. The mechanism by which extracellular signals are transmitted into the cell through conformational changes in Tar is predicted to involve a piston displacement of one of the α4 helices of the homodimer. To understand the molecular mechanisms underlying the induction of Tar activity by an attractant, the three-dimensional structures of the E. coli Tar periplasmic domain with and without bound aspartate, Asp-Tar and apo-Tar, respectively, were determined. Of the two ligand-binding sites, only one site was occupied, and it clearly showed the electron density of an aspartate. The slight changes in conformation and the electrostatic surface potential around the aspartate-binding site were observed. In addition, the presence of an aspartate stabilized residues Phe-150' and Arg-73. A pistonlike displacement of helix α4b' was also induced by aspartate binding as predicted by the piston model. Taken together, these small changes might be related to the induction of Tar activity and might disturb binding of the second aspartate to the second binding site in E. coli. PMID:27292793

  17. Quantitative autoradiography of [3H]CTOP binding to mu opioid receptors in rat brain

    [3H]H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ([3H]CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with [3H]CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited [3H]CTOP binding with high affinity (IC50 values of 0.2-2.4nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69,593 were very weak inhibitors of [3H]CTOP binding. Light microscopic autoradiography of [3H]CTOP binding sites revealed regions of high density and regions of moderate labeling. The cerebral cortex showed a low density of [3H]CTOP binding

  18. Analogues of doxanthrine reveal differences between the dopamine D 1 receptor binding properties of chromanoisoquinolines and hexahydrobenzo[a]phenanthridines

    Cueva, J.P.; Chemel, B.R.; Juncosa, J.I., Jr.; Lill, M.A.; Watts, V.J.; Nichols, D.E.

    2012-01-01

    Efforts to develop selective agonists for dopamine D 1-like receptors led to the discovery of dihydrexidine and doxanthrine, two bioisosteric ??-phenyldopamine-type full agonist ligands that display selectivity and potency at D 1-like receptors. We report herein an improved methodology for the synthesis of substituted chromanoisoquinolines (doxanthrine derivatives) and the evaluation of several new compounds for their ability to bind to D 1- and D 2-like receptors. Identical pendant phenyl ring substitutions on the dihydrexidine and doxanthrine templates surprisingly led to different effects on D 1-like receptor binding, suggesting important differences between the interactions of these ligands with the D 1 receptor. We propose, based on the biological results and molecular modeling studies, that slight conformational differences between the tetralin and chroman-based compounds lead to a shift in the location of the pendant ring substituents within the receptor. ?? 2011 Elsevier Ltd. All rights reserved.

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

    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. PMID:23954806

  20. Prediction on the binding domain between human interleukin-6 and its receptor

    冯健男; 任蕴芳; 沈倍奋

    2000-01-01

    Based on the spatial conformations of human interleukin-6 (hlL-6) derived from nuclear magnetic resonance analysis and human interleukin-6 receptor (hlL-6R) modeled with homology modeling method using human growth hormone receptor as template, the interaction between hlL-6 and its receptor (hIL-6R) is studied with docking program according to the surface electrostatic potential analysis and spatial conformation complement. The stable region structure composed of hlL-6 and hlL-6R is obtained on the basis of molecular mechanism optimization and molecular dynamics simulation. The binding domain between hIL-6 and hIL-6R is predicted theoretically. Furthermore, the especial binding sites that influence the interaction between hlL-6 and hlL-6R are confirmed. The results lay a theoretical foundation for confirming the active regions of hlL-6 and designing novel antagonist with computer-guided techniques.

  1. Prediction on the binding domain between human interleukin-6 and its receptor

    2000-01-01

    Based on the spatial conformations of human interleukin-6 (hIL-6) derived from nuclear magnetic resonance analysis and human interleukin-6 receptor (hIL-6R) modeled with homology modeling method using human growth hormone receptor as template, the interaction between hIL-6 and its receptor (hIL-6R) is studied with docking program according to the surface electrostatic potential analysis and spatial conformation complement. The stable region structure composed of hIL-6 and hIL-6R is obtained on the basis of molecular mechanism optimization and molecular dynamics simulation. The binding domain between hIL-6 and hIL-6R is predicted theoretically. Furthermore, the especial binding sites that influence the interaction between hIL-6 and hIL-6R are confirmed. The results lay a theoretical foundation for confirming the active regions of hIL-6 and designing novel antagonist with computer-guided techniques.

  2. Receptor binding assay technique for harmful algal bloom toxins quantification: reference manual

    The production of reference manual of procedures for distribution to laboratories engaged in shellfish toxicity research and monitoring activities and this manual is a contribution to the regional project which was funded by the Philippine Department of Science and Technology and produced by the Philippine Nuclear Research Institute. The main objectives of the project are the transfer of the receptor assay technology to interested Member States and the establishment of the receptor assay method in these laboratories and these objectives address the need for a rapid and sensitive assay to complement and replace the current live mouse bioassay in order to enhance shellfish toxicity monitoring program and improve the management of harmful algal blooms (HABs). Towards these objectives, a Regional (RCA) Training Workshop on Receptor Binding Assay Techniques for Harmful Algal Bloom Toxins Quantification was conducted at the Philippine Nuclear Research Institute (PNRI) from 29 Nov - 04 Dec 1999 and it aimed to train the RCA Member States representatives in neuroreceptor binding assays for paralytic shellfish poisoning (PSP) toxins as an alternative method to mouse bioassay

  3. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 1010 M-1, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r2>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG

  4. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  5. Hormone- and DNA-binding mechanisms of the recombinant human estrogen receptor.

    Obourn, J D; Koszewski, N J; Notides, A C

    1993-06-22

    We have investigated the hormone- and DNA-binding mechanisms of the wild-type human estrogen receptor (hER) overproduced in insect cells using a baculovirus expression system. The recombinant hER was indistinguishable in size (67 kDa) and immunogenically from the native human estrogen receptor in MCF-7 breast carcinoma cells. The recombinant hER was purified to 70-80% homogeneity with a two-step procedure that included ammonium sulfate precipitation and oligonucleotide affinity chromatography using a unique Teflon affinity matrix. The recombinant hER bound estradiol with a positively cooperative mechanism. At hER concentrations in excess of 13 nM the Hill coefficient reached a maximal value of 1.6, whereas, at lower hER concentrations, the Hill coefficient approached 1.0, suggesting that the hER was dissociated to the monomeric species and site-site interactions were diminished. The hER specifically bound an estrogen responsive element (ERE) from chicken vitellogenin II gene as measured by the gel mobility assay, ethylation, and thymine interference footprinting. Specific interference patterns suggest a two-fold symmetry of the hER binding to the ERE with each monomer of the hER bound in the major groove of the DNA. These data indicate that the recombinant hER is valuable to define the biochemical and structural properties of the native estrogen receptor. PMID:8512933

  6. Calcitonin receptor binding in the hen anterior pituitary during an oviposition cycle.

    Nakayama, Hiroyuki; Takahashi, Tetsuya; Nakagawa-Mizuyachi, Kaori; Kawashima, Mitsuo

    2011-10-01

    The equilibrium dissociation constant (K(d) ) and the maximum binding capacity (B(max) ) of calcitonin (CT) receptor in the plasma membrane of the anterior pituitary in hens were examined by Scatchard analysis of specific binding of (125) I-labeled chicken CT. Values of K(d) and B(max) of CT receptor were smaller in laying hens than in non-laying hens. A decrease in the K(d) and B(max) value of CT receptor was observed in the anterior pituitary after the injection of estradiol-17β and progesterone into nonlaying hens, but not changed after the injection of 5α-dihydrotestosterone. During an oviposition cycle, the K(d) and the B(max) value decreased 3 h before oviposition. In non-laying hens, neither the K(d) nor the B(max) value changed during a full day period. The present study suggests that the CT action on the anterior pituitary may increase 3 h before oviposition by the effect of estradiol-17β and progesterone in laying hens. PMID:21951904

  7. Synthesis, Nicotinic Acetylcholine Receptor Binding, and Antinociceptive Properties of 3′-(Substituted Phenyl)epibatidine Analogues. Nicotinic Partial Agonists⊥

    Carroll, F. Ivy; Ma, Wei; Deng, Liu; Navarro, Hernán A.; Damaj, M. Imad; Martin, Billy R.

    2010-01-01

    In 1992, John Daly et al. reported the isolation and structure determination of epibatidine. Epibatidine’s unique structure and its potent nicotinic agonist activity have had a tremendous impact on nicotine receptor research. This research has led to a better understanding of the nicotinic acetylcholine receptor (nAChR) pharmacophore and to epibatidine analogues with potential as pharmacotherapies for treating various CNS disorders. In this study, we report the synthesis, receptor binding ([3...

  8. DNA target selectivity by the vitamin D3 receptor: mechanism of dimer binding to an asymmetric repeat element.

    Towers, T L; Luisi, B F; Asianov, A; Freedman, L P

    1993-01-01

    The 1,25-dihydroxyvitamin D3 receptor, like other members of the nuclear receptor superfamily, forms dimers in solution that are probably stabilized by a dyad symmetrical interface formed by the ligand-binding domain. This receptor, however, recognizes DNA targets that are not dyad symmetric but rather are organized as direct repeats of a hexameric sequence with a characteristic 3-bp spacing. Using molecular modeling and site-directed mutagenesis, we have identified regions within the vitamin...

  9. Structure of the active core of human stem cell factor and analysis of binding to its receptor Kit

    Jiang, Xuliang; Gurel, Ogan; Mendiaz, Elizabeth A.; Stearns, George W.; Clogston, Christi L.; Lu, Hsieng S; Osslund, Timothy D.; Syed, Rashid S.; Langley, Keith E.; Hendrickson, Wayne A

    2000-01-01

    Stem cell factor (SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane-bound forms. It transduces signals by ligand- mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand-binding p...

  10. Binding of phylogenetically distant Bacillus thuringiensis cry toxins to a Bombyx mori aminopeptidase N suggests importance of Cry toxin's conserved structure in receptor binding.

    Shinkawa, A; Yaoi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-07-01

    We investigated the binding proteins for three Cry toxins, Cry1Aa, Cry1Ac, and the phylogenetically distant Cry9Da, in the midgut cell membrane of the silkworm. In a ligand blot experiment, Cry1Ac and Cry9Da bound to the same 120-kDa aminopeptidase N (APN) as Cry1Aa. A competition experiment with the ligand blot indicated that the three toxins share the same binding site on several proteins. The values of the dissociation constants of the three Cry toxins and 120-kDa APN are as low as the case of other Cry toxins and receptors. These results suggest that distantly related Cry toxins bind to the same site on the same proteins, especially with APN. We propose that the conserved structure in these three toxins includes the receptor-binding site. PMID:10387111

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

    Kvetny, J; Matzen, L E; Blichert-Toft, M; Watt-Boolsen, S; Date, J

    1989-01-01

    . u. to 94 a. u. and SHBG from 80 nmol/l to 69 nmol/l (P less than 0.05) followed after 6 weeks by a rise in serum TSH from 1.2 mU/l to 11.0 mU/l (P less than 0.05) suggesting an initial slight hypothyroidism. Nuclear receptor-binding of T4 and T3 increased within one week and eventually decreased to...... preresectional values. We conclude that the expected alteration of the metabolic state caused by resection of the gland is opposed by increased nuclear binding of T4 and T3.......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...

  12. The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

    Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

    2014-12-01

    The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

  13. Identification of two potential receptor-binding sites for hGM-CSF

    Eberhardt M.O.

    2003-01-01

    Full Text Available Two receptor-binding sites for hGM-CSF are described. Competitive binding ELISA using four monoclonal antibodies (MAbs showed different epitope recognitions. The antibody combining sites were mapped using sets of overlapping peptides and hexapeptide libraries prepared by the SPOT synthesis technique. We identified the conformationally dependent epitopes A18E21R23R24F119 and R23E21N17W13 bound by MAb CC5B5 and the nonlinear epitope P118F119W13E14 bound by MAb M1B8. The epitopes recognized by these two MAbs are very closely located on the native protein surface. The peptide L61YKQGKLRGSLTK72 was recognized by MAb M7E10 and the peptide A1PAR4, representing the N-terminal sequence of the protein, was bound by the nonneutralizing MAb CC1H7. Inhibition assays of the GM-CSF biological activity demonstrated that MAb M1B8, CC5B5 and M7E10 bind to domains which are responsible for the interaction of the cytokine with the GM-CSF receptor.

  14. Mu Opioid Receptor Binding Correlates with Nicotine Dependence and Reward in Smokers.

    Hiroto Kuwabara

    Full Text Available The rewarding effects of nicotine are associated with activation of nicotine receptors. However, there is increasing evidence that the endogenous opioid system is involved in nicotine's rewarding effects. We employed PET imaging with [11C]carfentanil to test the hypotheses that acute cigarette smoking increases release of endogenous opioids in the human brain and that smokers have an upregulation of mu opioid receptors (MORs when compared to nonsmokers. We found no significant changes in binding potential (BPND of [11C]carfentanil between the placebo and the active cigarette sessions, nor did we observe differences in MOR binding between smokers and nonsmokers. Interestingly, we showed that in smokers MOR availability in bilateral superior temporal cortices during the placebo condition was negatively correlated with scores on the Fagerström Test for Nicotine Dependence (FTND. Also in smokers, smoking-induced decreases in [11C]carfentanil binding in frontal cortical regions were associated with self-reports of cigarette liking and wanting. Although we did not show differences between smokers and nonsmokers, the negative correlation with FTND corroborates the role of MORs in superior temporal cortices in nicotine addiction and provides preliminary evidence of a role of endogenous opioid signaling in frontal cortex in nicotine reward.

  15. Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

    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.

  16. Momordica charantia and its novel polypeptide regulate glucose homeostasis in mice via binding to insulin receptor.

    Lo, Hsin-Yi; Ho, Tin-Yun; Lin, Chingju; Li, Chia-Cheng; Hsiang, Chien-Yun

    2013-03-13

    Momordica charantia (MC) has been used as an alternative therapy for diabetes mellitus. This study analyzed and elucidated therapeutic targets contributing to the hypoglycemic effect of aqueous extract of MC seeds (MCSE) by transcriptomic analysis. Protein ingredients aimed at the hypoglycemic target were further identified by proteomic, docking, and receptor-binding assays. The data showed that MSCE (1 g/kg) significantly lowered the blood glucose level in normal and diabetic mice. Moreover, MCSE primarily regulated the insulin signaling pathway in muscles and adipose tissues, suggesting that MCSE might target insulin receptor (IR), stimulate the IR-downstream pathway, and subsequently display hypoglycemic activity in mice. It was further revealed that inhibitor against trypsin (TI) of MC directly docked into IR and activated the kinase activity of IR in a dose-dependent manner. In conclusion, the findings suggested that MCSE regulated glucose metabolism mainly via the insulin signaling pathway. Moreover, TI was newly identified as a novel IR-binding protein of MC that triggered the insulin signaling pathway via binding to IR. PMID:23414136

  17. Genomic analysis of hepatic Farnesoid X Receptor (FXR) binding sites reveals altered binding in obesity and direct gene repression by FXR

    Lee, Jiyoung; Seok, Sun Mi; Yu, Pengfei; Kim, Kyungsu; Smith, Zachary; Rivas-Astroza, Marcelo; Zhong, Sheng; Kemper, Jongsook Kim

    2012-01-01

    The nuclear bile acid receptor, Farnesoid X Receptor (FXR), is an important transcriptional regulator of liver metabolism. Despite recent advances in understanding its functions, how FXR regulates genomic targets and whether the transcriptional regulation by FXR is altered in obesity remain largely unknown. Here, we analyzed hepatic genome-wide binding sites of FXR in normal and dietary obese mice by chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. A total of 15,263 and 5,272 FXR...

  18. In Silico Investigation of the Neurotensin Receptor 1 Binding Site: Overlapping Binding Modes for Small Molecule Antagonists and the Endogenous Peptide Agonist.

    Lückmann, Michael; Holst, Birgitte; Schwartz, Thue W; Frimurer, Thomas M

    2016-01-01

    The neurotensin receptor 1 (NTSR1) belongs to the family of 7TM, G protein-coupled receptors, and is activated by the 13-amino-acid peptide neurotensin (NTS) that has been shown to play important roles in neurological disorders and the promotion of cancer cells. Recently, a high-resolution x-ray crystal structure of NTSR1 in complex with NTS8-13 has been determined, providing novel insights into peptide ligand recognition by 7TM receptors. SR48692, a potent and selective small molecule antagonist has previously been used extensively as a tool compound to study NTSR1 receptor signaling properties. To investigate the binding mode of SR48692 and other small molecule compounds to NTSR1, we applied an Automated Ligand-guided Backbone Ensemble Receptor Optimization protocol (ALiBERO), taking receptor flexibility and ligand knowledge into account. Structurally overlapping binding poses for SR48692 and NTS8-13 were observed, despite their distinct chemical nature and inverse pharmacological profiles. The optimized models showed significantly improved ligand recognition in a large-scale virtual screening assessment compared to the crystal structure. Our models provide new insights into small molecule ligand binding to NTSR1 and could facilitate the structure-based design of non-peptide ligands for the evaluation of the pharmacological potential of NTSR1 in neurological disorders and cancer. PMID:27491650

  19. Expression of HIV receptors, alternate receptors and co-receptors on tonsillar epithelium: implications for HIV binding and primary oral infection

    Maher Diane M

    2006-04-01

    Full Text Available Abstract Background Primary HIV infection can develop from exposure to HIV in the oral cavity. In previous studies, we have documented rapid and extensive binding of HIV virions in seminal plasma to intact mucosal surfaces of the palatine tonsil and also found that virions readily penetrated beneath the tissue surfaces. As one approach to understand the molecular interactions that support HIV virion binding to human mucosal surfaces, we have examined the distribution of the primary HIV receptor CD4, the alternate HIV receptors heparan sulfate proteoglycan (HS and galactosyl ceramide (GalCer and the co-receptors CXCR4 and CCR5 in palatine tonsil. Results Only HS was widely expressed on the surface of stratified squamous epithelium. In contrast, HS, GalCer, CXCR4 and CCR5 were all expressed on the reticulated epithelium lining the tonsillar crypts. We have observed extensive variability, both across tissue sections from any tonsil and between tonsils, in the distribution of epithelial cells expressing either CXCR4 or CCR5 in the basal and suprabasal layers of stratified epithelium. The general expression patterns of CXCR4, CCR5 and HS were similar in palatine tonsil from children and adults (age range 3–20. We have also noted the presence of small clusters of lymphocytes, including CD4+ T cells within stratified epithelium and located precisely at the mucosal surfaces. CD4+ T cells in these locations would be immediately accessible to HIV virions. Conclusion In total, the likelihood of oral HIV transmission will be determined by macro and micro tissue architecture, cell surface expression patterns of key molecules that may bind HIV and the specific properties of the infectious inoculum.

  20. Iron Uptake Mediated by Binding of H-Ferritin to the TIM-2 Receptor in Mouse Cells

    Han, Jian; William E. Seaman; Di, Xiumin; Wang, Wei; Willingham, Mark; Torti, Frank M.; Torti, Suzy V.

    2011-01-01

    Ferritin binds specifically and saturably to a variety of cell types, and recently several ferritin receptors have been cloned. TIM-2 is a specific receptor for H ferritin (HFt) in the mouse. TIM-2 is a member of the T cell immunoglobulin and mucin domain containing (TIM) protein family and plays an important role in immunity. The expression of TIM-2 outside of the immune system indicates that this receptor may have broader roles. We tested whether ferritin binding to TIM-2 can serve as an ir...

  1. Specific binding of a ligand of σ-opioid receptors - N-allylnormetazocine (SKF 10047) - with liver membranes

    A ligand of the σ-opioid receptors - N-allylnormetazocine (SKF 10047) -binds specifically and reversible with rat liver membranes. In relation to a number of properties, the sites binding SKF 10047 in the liver are similar to the σ-opioid receptors of the central nervous system. They do not interact with classical opiates (morphine, naloxone) and with opioid peptides, but bind well benzomorphans (bremazocine, SKF 10047) and a number of compounds of different chemical structures with a pronounced psychtropic action (haloperidol, imipramine, phencyclidine, etc.)

  2. Synthetic Peptide Ligands of the Antigen Binding Receptor Induce Programmed Cell Death in a Human B-Cell Lymphoma

    Renschler, Markus F.; Bhatt, Ramesh R.; Dower, William J.; Levy, Ronald

    1994-04-01

    Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.

  3. Specific binding of a ligand of sigma-opioid receptors - N-allylnormetazocine (SKF 10047) - with liver membranes

    Samovilova, N.N.; Yarygin, K.N.; Vinogradov, V.A.

    1986-08-01

    A ligand of the sigma-opioid receptors - N-allylnormetazocine (SKF 10047) -binds specifically and reversible with rat liver membranes. In relation to a number of properties, the sites binding SKF 10047 in the liver are similar to the sigma-opioid receptors of the central nervous system. They do not interact with classical opiates (morphine, naloxone) and with opioid peptides, but bind well benzomorphans (bremazocine, SKF 10047) and a number of compounds of different chemical structures with a pronounced psychtropic action (haloperidol, imipramine, phencyclidine, etc.).

  4. Identification of the estrogen receptor Cd-binding sites by chemical modification.

    Nesatyy, Victor J; Rutishauser, Barbara V; Eggen, Rik I L; Suter, Marc J-F

    2005-07-01

    The widely reported interactions of the estrogen receptor (ER) with endocrine disrupting chemicals (EDCs) present in the environment gave raise to public concern and led to a number of screening and testing initiatives on the international level. Recent studies indicated that certain heavy metals, including cadmium, can mimic the effects of the endogenous estrogen receptor agonist 17beta-estradiol, and lead to estrogen receptor activation. Previous studies of the chimeric proteins, which incorporate the ligand-binding domain of the human ER, identified Cys 381, Cys 447, Glu 523, His 524 and Asp 538 as possible sites of interactions with cadmium. In the present study we utilized the rainbow trout ER ligand-binding domain fused to glutathione-S-transferase, and used Cd-shielding against various types of chemical modification of the fusion protein to study non-covalent interactions between the ER and Cd. The distribution of exposed and shielded residues allowed to identify amino acid residues involved in the interaction. Our data indicated preferential protection of Cys groups by cadmium, suggesting their involvement in the interaction. This supports data found in the literature on the strong binding affinity of the thiol group towards metals. However, not all Cys in the fusion protein sequence were protected against chemical modification, illustrating the importance of their chemical environment. In general, the location of rtER-LBD Cys residues implicated in Cd interactions did not confirm assignments made by alanine-scanning mutagenesis for the hER, probably due to differences in experimental setup and fusion proteins used. The involvement of other functional groups such as carboxylic acids in the Cd interactions, though not confirmed, can not be completely ruled out due to the general limitations of the chemical modification approach discussed in detail. Suggestions for an improved experimental setup were made. PMID:15965534

  5. Sensitivity of binding of high-affinity dopamine receptor radioligands to increased synaptic dopamine.

    Gatley, S J; Gifford, A N; Carroll, F I; Volkow, N D

    2000-12-15

    PET and SPECT studies have documented that D2 radioligands of moderate affinity, but not radioligands of high affinity, are sensitive to pharmacological challenges that alter synaptic dopamine levels. The objective of this work was to determine whether the brain kinetics of high-affinity radioligands for dopamine D1 ([(3)H]SCH 23390) and D2 ([(123)I]epidepride) receptors were altered by a prolonged elevation of synaptic dopamine induced by the potent cocaine analog RTI-55. Mice were injected intravenously with radioligands either 30 min after or 4 h before intraperitoneal administration of RTI-55 (2 mg/kg). In separate experiments, the pharmacological effects of RTI-55 were assessed biochemically by measuring uptake of dopamine in synaptosomes prepared from RTI-treated mice and behaviorally by monitoring locomotor activity. Consistent with the expected elevation of synaptic dopamine, RTI-55 induced a long-lasting decrement in dopamine uptake measured ex vivo, and a prolonged increase in locomotor activity. RTI-55 injected prior to the radioligands induced a significant (P epidepride at 15 min, relative to saline-treated controls, but there were no differences between the two groups at later time-points. For [(3)H]SCH 23390, both initial striatal uptake and subsequent clearance were slightly increased by preadministration of RTI-55. Administration of RTI-55 4 h after the radioligands (i.e., when it was presumed that a state of near equilibrium binding of the radioligands had been reached), was associated with a significant reduction of striatal radioactivity for both radiotracers. Our results are consistent with increased competition between dopamine and radioligand for binding to both D1 and D2 receptors after treatment with RTI-55. We suggest that the magnitude of the competition is reduced by failure of the receptor binding of high-affinity radioligands to rapidly attain equilibrium. PMID:11044896

  6. Truncated atrial natriuretic peptide (ANP) analogs: relationship between receptor binding and cyclic GMP accumulation

    The authors have shown that cultured bovine aortic smooth muscle (BASM) cells contain high affinity receptor sites for the ANP, auriculin (ANP(4-28)). Furthermore, ANP(4-28) causes cyclic GMP (cGMP) levels to increase in BASM. In the present study, the authors synthesized a series of NH2 and/or COOH truncated ANP(4-28) analogs and examined their ability to compete for 125I-ANP(4-28) binding to BASM and increase cGMP levels. ANP-mediated cGMP responses were reduced when amino acids were deleted from the NH2 and/or COOH termini of ANP(4-28). Removal of the NH2 terminal R, R-S, or R-S-S residues resulted in a 10X decrease in potency for cGMP stimulation. Deletion of the COOH terminal R-Y and F-R-Y residues resulted in a marked decline in potency. ANP's lacking the F-R-Y tripeptide were nearly inactive in stimulating cGMP accumulation. In contrast to the cGMP effects, NH2 and/or COOH truncations of ANP(4-28) did not alter apparent receptor binding affinities (Ki(app)). All of these peptide analogs exhibited Ki(app)'s of 1-5 nM. Furthermore, peptides that bound effectively and failed to elicit cGMP responses did not antagonize ANP(4-28)-mediated cGMP increases. These binding and functional data suggest the presence of a single class of ANP receptors on BASM is insufficient to explain the actions of ANP's in these cells

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

    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.

  8. 3D Pharmacophore, hierarchical methods, and 5-HT4 receptor binding data.

    Varin, Thibault; Saettel, Nicolas; Villain, Jonathan; Lesnard, Aurelien; Dauphin, François; Bureau, Ronan; Rault, Sylvain

    2008-10-01

    5-Hydroxytryptamine subtype-4 (5-HT(4)) receptors have stimulated considerable interest amongst scientists and clinicians owing to their importance in neurophysiology and potential as therapeutic targets. A comparative analysis of hierarchical methods applied to data from one thousand 5-HT(4) receptor-ligand binding interactions was carried out. The chemical structures were described as chemical and pharmacophore fingerprints. The definitions of indices, related to the quality of the hierarchies in being able to distinguish between active and inactive compounds, revealed two interesting hierarchies with the Unity (1 active cluster) and pharmacophore fingerprints (4 active clusters). The results of this study also showed the importance of correct choice of metrics as well as the effectiveness of a new alternative of the Ward clustering algorithm named Energy (Minimum E-Distance method). In parallel, the relationship between these classifications and a previously defined 3D 5-HT(4) antagonist pharmacophore was established. PMID:18821249

  9. Mutational analysis of the putative receptor-binding domain of Moloney murine leukemia virus glycoprotein gp70.

    Panda, B R; Kingsman, S M; Kingsman, A J

    2000-07-20

    The entry of Moloney murine leukemia virus (MoMuLV) to murine cells is mediated by the binding of its envelope glycoprotein gp70 to its receptor, the cationic amino acid transporter MCAT-1. The binding property of the envelope protein lies mainly in the N-terminal half of the protein. To identify essential residues involved in the binding of gp70 to its receptor, we have mutated amino acids within the putative receptor-binding domain of MoMuLV gp70. Changes in the residues P94 and W100 resulted in lower viral titers in comparison to the wild-type virions. Single, double, or triple point mutations involving the residue W100 make the envelope protein severely defective in binding to its receptor. Binding studies and cell fusion experiments with murine XC cells suggested that the residue W100 might play an important role in the process of infection by making contact between gp70 and its receptor. PMID:10891411

  10. Production and characterization of chimeric transferrins for the determination of the binding domains for bacterial transferrin receptors.

    Retzer, M D; Kabani, A; Button, L L; Yu, R H; Schryvers, A B

    1996-01-12

    Pathogenic bacteria in the Neisseriaceae and Pasteurellaceae possess outer membrane proteins that specifically bind transferrin from the host as the first step in the iron acquisition process. As a logical progression from prior studies of the ligand-receptor interaction using biochemical approaches, we have initiated an approach involving the production of recombinant chimeric transferrins to further identify the regions of transferrin involved in receptor binding. In order to prepare bovine/human hybrids, the bovine transferrin gene was cloned, sequenced, and compared with the existing human transferrin gene sequence. After identification of potential splice sites, hybrid transferrin genes were constructed using the polymerase chain reaction-based approach of splicing by overlap extension. Five hybrid genes containing sequences from both bovine and human transferrin were constructed. Recombinant transferrins were produced in a baculovirus expression vector system and affinity-purified using concanavalin A-Sepharose. The recombinant proteins were analyzed for reactivity against polyclonal and monoclonal antibodies and assessed for binding to Neisseria meningitidis transferrin receptor proteins in solid-phase binding assays and affinity isolation experiments. These experiments enabled us to localize the regions of human transferrin predominantly involved in binding to the N. meningitidis receptor to amino acid residues 346-588. The construction of these chimeras provides unique tools for the investigation of transferrin binding to receptors from both human and bovine bacterial pathogens. PMID:8557646

  11. Thyroid hormone receptor binds to a site in the rat growth hormone promoter required for induction by thyroid hormone

    Transcription of the rat growth hormone (rGH) gene in pituitary cells is increased by addition of thyroid hormone (T3). This induction is dependent on the presence of specific sequences just upstream of the rGH promoter. The authors have partially purified T3 receptor from rat liver and examined its interaction with these rGH sequences. They show here that T3 receptor binds specifically to a site just upstream of the basal rGH promoter. This binding site includes two copies of a 7-base-pair direct repeat, the centers of which are separated by 10 base pairs. Deletions that specifically remove the T3 receptor binding site drastically reduce response to T3 in transient transfection experiments. These results demonstrate that T3 receptor can recognize specific DNA sequences and suggest that it can act directly as a positive transcriptional regulatory factor

  12. Localization of the binding site for the human high-affinity Fc receptor on IgG.

    Duncan, A R; Woof, J M; Partridge, L J; Burton, D R; Winter, G

    1988-04-01

    A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen. PMID:2965792

  13. Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity.

    Nguyen, Duy P; Miyaoka, Yuichiro; Gilbert, Luke A; Mayerl, Steven J; Lee, Brian H; Weissman, Jonathan S; Conklin, Bruce R; Wells, James A

    2016-01-01

    Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants. PMID:27363581

  14. Binding of ArgTX-636 in the NMDA receptor ion channel

    Poulsen, Mette H; Andersen, Jacob; Christensen, Rune;

    2015-01-01

    N1 and GluN2A where mutation influences ArgTX-636 potency suggests differential contribution of the M2-loops of GluN1 and GluN2A to binding of ArgTX-636. The results of the mutational analysis are highly relevant for the future structure-based development of argiotoxin-derived NMDAR channel blockers.......The N-methyl-d-aspartate receptors (NMDARs) constitute an important class of ligand-gated cation channels that are involved in the majority of excitatory neurotransmission in the human brain. Compounds that bind in the NMDAR ion channel and act as blockers are use- and voltage-dependent inhibitors...... of NMDAR activity and have therapeutic potential for treatment of a variety of brain diseases or as pharmacological tools for studies of the neurobiological role of NMDARs. We have performed a kinetic analysis of the blocking mechanism of the prototypical polyamine toxin NMDAR ion channel blocker...

  15. Metal ion enhanced binding of AMD3100 to Asp262 in the CXCR4 receptor

    Gerlach, Lars Ole; Jakobsen, Janus S; Jensen, Kasper P; Rosenkilde, Mette R; Skerlj, Renato T; Ryde, Ulf; Bridger, Gary J; Schwartz, Thue W

    2003-01-01

    The affinity of AMD3100, a symmetrical nonpeptide antagonist composed of two 1,4,8,11-tetraazacyclotetradecane (cyclam) rings connected through a 1,4-dimethylene(phenylene) linker to the CXCR4 chemokine receptor was increased 7, 36, and 50-fold, respectively, by incorporation of the following: Cu(2......+), Zn(2+), or Ni(2+) into the cyclam rings of the compound. The rank order of the transition metal ions correlated with the calculated binding energy between free acetate and the metal ions coordinated in a cyclam ring. Construction of AMD3100 substituted with only a single Cu(2+) or Ni(2+) ion...... of the bicyclam is dependent on both Asp(171) and Asp(262), the enhancing effect of the metal ion was selectively eliminated by substitution of Asp(262) located at the extracellular end of TM-VI. It is concluded that the increased binding affinity of the metal ion substituted AMD3100 is obtained...

  16. Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity

    Nguyen, Duy P.; Miyaoka, Yuichiro; Gilbert, Luke A.; Mayerl, Steven J.; Lee, Brian H.; Weissman, Jonathan S.; Conklin, Bruce R.; Wells, James A.

    2016-01-01

    Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants. PMID:27363581

  17. Chronic ACE inhibitor treatment increases angiotensin type 1 receptor binding in vivo in the dog kidney

    PET imaging has been recently introduced for investigating the type 1 angiotensin II receptor (AT1R) in vivo. The goal of the present study was to investigate the effects of acute and chronic exposure to angiotensin converting enzyme inhibitors (ACEI) on the AT1R in the dog kidney. Animals were imaged at baseline, after acute intravenous ACEI treatment and after a chronic 2-week exposure to an oral ACEI. Control animals were imaged at identical time points in the absence of ACEI treatment. In vivo AT1R binding expressed by Ki was increased in the renal cortex by chronic ACEI treatment (p 1R density (Bmax) also revealed significant increases in AT1R in isolated glomeruli (p 1R binding in vivo in the dog renal cortex. (orig.)

  18. Contribution of a p75 interleukin 2 binding peptide to a high-affinity interleukin 2 receptor complex

    There are at least two forms of cellular receptors for interleukin 2(IL-2); one with a very high affinity and the other with a lower affinity. The authors identified a non-Tac IL-2 binding peptide with a relative molecular weight of 75,000 (p75). Cell lines bearing either the p55 Tac or the p75 peptide alone manifested low-affinity IL-2 binding, whereas a cell line bearing both peptides manifested both high- and low-affinity receptors. Cross-linking studies were performed with 125I-labeled IL-2. After the internalization of labeled IL-2 through high-affinity receptors, the p75 peptide could not be detected by cross-linking studies. Furthermore, fusion of cell membranes from low-affinity IL-2 binding cell lines bearing the Tac peptide alone with membranes from a cell line bearing the p75 peptide alone generated hybrid membranes bearing high-affinity receptors. These results suggest a multichain model for the high-affinity Il-2 receptor in which high-affinity receptors would be expressed when both Tac and p75 Il-2 binding peptides are present and associated in a receptor complex

  19. Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding

    Broghammer, Angelique; Krusell, Lene; Blaise, Mickaël;

    2012-01-01

    , and NFR1 protein retained its in vitro kinase activity. Processing of NFR5 protein was characterized by determining the N-glycosylation patterns of the ectodomain. Two different glycan structures with identical composition, Man(3)XylFucGlcNAc(4), were identified by mass spectrometry and located at...... amino acid positions N68 and N198. Receptor-ligand interaction was measured by using ligands that were labeled or immobilized by application of chemoselective chemistry at the anomeric center. High-affinity ligand binding was demonstrated with both solid-phase and free solution techniques. The K...

  20. Synthesis, Nicotinic Acetylcholine Receptor Binding, and Pharmacological Properties of 3’- (Substituted phenyl) Deschloroepibatidine Analogs

    F. Ivy Carroll; Yokota, Yasuno; Ma, Wei; Lee, Jeffrey R.; Brieaddy, Lawrence E.; Burgess, Jason P.; Navarro, Hernán A.; Damaj, M. I.; Martin, Billy R.

    2007-01-01

    A series of 3’-(substituted phenyl)deschloroepibatidine analogs (5a–j) were synthesized. The α4β2* and α7 nicotinic acetylcholine receptor (nAChR) binding properties and functional activity in the tail-flick, hot-plate, locomotor, and body temperature tests in mice of 5a–j were compared to those of the nAChR agonist, nicotine (1), epibatidine (4), and deschloroepibatidine (13) the partial agonist, varenicline (3) and the antagonist 2’-fluoro-3’-(substituted phenyl)deschloroepibatidine analogs...

  1. Role of receptor binding specificity in influenza A virus transmission and pathogenesis

    De Graaf, M.; Fouchier, R. A. M.

    2014-01-01

    The recent emergence of a novel avian A/H7N9 influenza virus in poultry and humans in China, as well as laboratory studies on adaptation and transmission of avian A/H5N1 influenza viruses, has shed new light on influenza virus adaptation to mammals. One of the biological traits required for animal influenza viruses to cross the species barrier that received considerable attention in animal model studies, in vitro assays, and structural analyses is receptor binding specificity. Sialylated glyc...

  2. Modulation of radioligand binding to the GABA(A)-benzodiazepine receptor complex by a new component from Cyperus rotundus.

    Ha, Jeoung-Hee; Lee, Kwang-Youn; Choi, Hyoung-Chul; Cho, Jungsook; Kang, Byung-Soo; Lim, Jae-Chul; Lee, Dong-Ung

    2002-01-01

    Four sesquiterpenes, beta-selinene, isocurcumenol, nootkatone and aristolone and one triterpene, oleanolic acid were isolated from the ethylacetate fraction of the rhizomes of Cyperus rotundus and tested for their ability to modulate gamma-aminobutyric acid (GABA(A))-benzodiazepine receptor function by radioligand binding assays using rat cerebrocortical membranes. Among these compounds, only isocurcumenol, one of the newly identified constituents of this plant, was found to inhibit [3H]Ro15-1788 binding and enhance [3H]flunitrazepam binding in the presence of GABA. These results suggest that isocurcumenol may serve as a benzodiazepine receptor agonist and allosterically modulate GABAergic neurotransmission via enhancement of endogenous receptor ligand binding. PMID:11824542

  3. Binding studies using Pichia pastoris expressed human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins.

    Zheng, Yujuan; Xie, Jinghang; Huang, Xin; Dong, Jin; Park, Miki S; Chan, William K

    2016-06-01

    The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR of other species, since it is relatively unstable and less sensitive to some ligands in vitro. Overexpression of human AHR has been limited to the baculovirus expression, which is costly and tedious due to the need of repetitive baculovirus production. Here we explored whether we could generate abundant amounts of human AHR and ARNT in a better overexpression system for functional study. We observed that human AHR and ARNT can be expressed in Pichia pastoris with yields that are comparable to the baculovirus system only if their cDNAs are optimized for Pichia expression. Fusion with a c-myc tag at their C-termini seems to increase the expression yield. These Pichia expressed proteins can effectively heterodimerize and form the ternary AHR/ARNT/enhancer complex in the presence of β-naphthoflavone or kynurenine. Limited proteolysis using thermolysin can be used to study the heterodimerization of these human AHR and ARNT proteins. PMID:26923060

  4. Dopaminergic receptor-ligand binding assays based on molecularly imprinted polymers on quartz crystal microbalance sensors.

    Naklua, Wanpen; Suedee, Roongnapa; Lieberzeit, Peter A

    2016-07-15

    Molecularly imprinted polymers (MIPs) have been successfully applied as selective materials for assessing the binding activity of agonist and antagonist of dopamine D1 receptor (D1R) by using quartz crystal microbalance (QCM). In this study, D1R derived from rat hypothalamus was used as a template and thus self-organized on stamps. Those were pressed into an oligomer film consisting of acrylic acid: N-vinylpyrrolidone: N,N'-(1,2-dihydroxyethylene) bis-acrylamide in a ratio of 2:3:12 spin coated onto a dual electrode QCM. Such we obtained one D1R-MIP-QCM electrode, whereas the other electrode carried the non-imprinted control polymer (NIP) that had remained untreated. Successful imprinting of D1R was confirmed by AFM. The polymer can re-incorporate D1R leading to frequency responses of 100-1200Hz in a concentration range of 5.9-47.2µM. In a further step such frequency changes proved inherently useful for examining the binding properties of test ligands to D1R. The resulting mass-sensitive measurements revealed Kd of dopamine∙HCl, haloperidol, and (+)-SCH23390 at 0.874, 25.6, and 0.004nM, respectively. These results correlate well with the values determined in radio ligand binding assays. Our experiments revealed that D1R-MIP sensors are useful for estimating the strength of ligand binding to the active single site. Therefore, we have developed a biomimetic surface imprinting strategy for QCM studies of D1R-ligand binding and presented a new method to ligand binding assay for D1R. PMID:26926593

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

    Xu, Minli; Su, Zhengchang

    2009-01-01

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

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

    Su Zhengchang

    2009-01-01

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

  7. Analysis of the Binding Sites of Porcine Sialoadhesin Receptor with PRRSV

    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.

  8. Inhibitory mechanisms and binding site location for serotonin selective reuptake inhibitors on nicotinic acetylcholine receptors.

    Arias, Hugo R; Feuerbach, Dominik; Bhumireddy, Pankaj; Ortells, Marcelo O

    2010-05-01

    Functional and structural approaches were used to examine the inhibitory mechanisms and binding site location for fluoxetine and paroxetine, two serotonin selective reuptake inhibitors, on nicotinic acetylcholine receptors (AChRs) in different conformational states. The results establish that: (a) fluoxetine and paroxetine inhibit h alpha1beta1 gammadelta AChR-induced Ca(2+) influx with higher potencies than dizocilpine. The potency of fluoxetine is increased approximately 10-fold after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]cytisine binding to resting but activatable Torpedo AChRs elicited by these antidepressants, (b) fluoxetine and paroxetine inhibit the binding of the phencyclidine analog piperidyl-3,4-(3)H(N)]-(N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine to the desensitized Torpedo AChR with higher affinities compared to the resting AChR, and (c) fluoxetine inhibits [(3)H]dizocilpine binding to the desensitized AChR, suggesting a mutually exclusive interaction. This is supported by our molecular docking results where neutral dizocilpine and fluoxetine and the conformer of protonated fluoxetine with the highest LUDI score interact with the domain between the valine (position 13') and leucine (position 9') rings. Molecular mechanics calculations also evidence electrostatic interactions of protonated fluoxetine at positions 20', 21', and 24'. Protonated dizocilpine bridges these two binding domains by interacting with the valine and outer (position 20') rings. The high proportion of protonated fluoxetine and dizocilpine calculated at physiological pH suggests that the protonated drugs can be attracted to the channel mouth before binding deeper within the AChR ion channel between the leucine and valine rings, a domain shared with phencyclidine, finally blocking ion flux and inducing AChR desensitization. PMID:20079457

  9. Heptapeptide ligands against receptor-binding sites of influenza hemagglutinin toward anti-influenza therapy.

    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. PMID:26833245

  10. 125I-iomazenil-benzodiazepine receptor binding during psychological stress in rats

    We investigated the changes in 125I-iomazenil (125I-IMZ) benzodiazepine receptor (BZR) binding with psychological stress in a rat model. Six male Wistar rats were placed under psychological stress for 1 hour by using a communication box. No physical stress was not received. 1.85 MBq of 125I-IMZ was injected into the lateral tail vein and the rat was killed 3 hours later. Twenty-micormeter-thick sections of the brain were collected and % injected dose per body weight (% ID/BW) of eleven regions (frontal, parietal, temporal, occipital cortices, caudate putamen, accumubens nuclei, globus pallidus, amygdala, thalamus, hippocampus and hypothalamus) were calculated by autoradiography. The %ID/BW of rats which were placed under psychological stress was compared with that of 6 control rats. The %ID/BW of rats which were placed under psychological stress diffusely tended to show a reduction in 125I-IMZ-BZR binding. A significant decrease in BZR binding was observed in the hippocampus of the rats which were placed under psychological stress. 125I-IMZ-BZR binding tended to decrease throughout the brain. (author)

  11. Identification of the bioactive and consensus peptide motif from Momordica charantia insulin receptor-binding protein.

    Lo, Hsin-Yi; Li, Chia-Cheng; Ho, Tin-Yun; Hsiang, Chien-Yun

    2016-08-01

    Many food bioactive peptides with diverse functions have been discovered by studying plant proteins. We have previously identified a 68-residue insulin receptor (IR)-binding protein (mcIRBP) from Momordica charantia that exhibits hypoglycemic effects in mice via interaction with IR. By in vitro digestion, we found that mcIRBP-19, spanning residues 50-68 of mcIRBP, enhanced the binding of insulin to IR, stimulated the phosphorylation of PDK1 and Akt, induced the expression of glucose transporter 4, and stimulated both the uptake of glucose in cells and the clearance of glucose in diabetic mice. Furthermore, mcIRBP-19 homologs were present in various plants and shared similar β-hairpin structures and IR kinase-activating abilities to mcIRBP-19. In conclusion, our findings suggested that mcIRBP-19 is a blood glucose-lowering bioactive peptide that exhibits IR-binding potentials. Moreover, we newly identified novel IR-binding bioactive peptides in various plants which belonged to different taxonomic families. PMID:26988505

  12. In vivo [3H]spiperone binding: evidence for accumulation in corpus striatum by agonist-mediated receptor internalization

    The processes of receptor internalization and recycling have been well-documented for receptors for hormones, growth factors, lysosomal enzymes, and cellular substrates. Evidence also exists that these processes also occur for beta-adrenergic, muscarinic cholinergic, and delta-opiate receptors in frog erythrocytes or cultured nervous tissue. In this study, evidence is presented that agonist-mediated receptor internalization and recycling occurs at the dopamine receptor in rat corpus striatum. First, the in vivo binding of the dopamine antagonist [3H]spiperone was increased by both electrical stimulation and pharmacologically induced increases of dopamine release. Conversely, depletion of dopamine with reserpine decreased in vivo [3H]spiperone binding, but the same reserpine treatment did not alter its in vitro binding. Second, the rate of dissociation of [3H]spiperone from microsomal membranes prepared from rat striatum following in vivo binding was fivefold slower than its dissociation following in vitro equilibrium binding. Mild detergent treatment, employed to disrupt endocytic vesicle membranes, increased the rate of dissociation of in vivo bound [3H]spiperone from microsomal membranes to values not significantly different from its in vitro bound dissociation rate. Third, treatment of rats with chloroquine, a drug that prevents receptor recycling but not internalization, prior to [3H]spiperone injection resulted in a selective increase of in vivo [3H]spiperone binding in the light microsome membranes. The existence of mechanisms that rapidly alter the number of neurotransmitter receptors at synapses provides dynamic regulation of receptors in response to varied acute stimulation states

  13. Receptor binding specificity of recent human H3N2 influenza viruses

    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

  14. Familial Risk for Major Depression is Associated with Lower Striatal 5-HT4 Receptor Binding

    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...... was to determine whether familial risk for MDD is associated with cerebral 5-HT4 receptor binding as measured with [(11)C]SB207145 brain PET imaging. Familial risk is the most potent risk factor of MDD. METHODS: We studied 57 healthy individuals (mean age 36 yrs, range 20-86; 21 women), 26 of which...... depression, and that lower striatal 5-HT4 receptor binding is associated with increased risk for developing MDD. The finding is intriguing considering that the 5-HT4 receptor has been suggested to be an effective target for antidepressant treatment....

  15. Redox modification of ryanodine receptors by mitochondria-derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts.

    Cooper, Leroy L; Li, Weiyan; Lu, Yichun; Centracchio, Jason; Terentyeva, Radmila; Koren, Gideon; Terentyev, Dmitry

    2013-12-01

    Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca(2+)) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca(2+) handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5-9 months) and old (4-6 years) rabbit hearts to test the hypothesis that changes in Ca(2+) homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) by mitochondria-derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca(2+) handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca(2+) dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age-related changes in ROS production and mitochondria membrane potential using a ROS-sensitive dye and a mitochondrial voltage-sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca(2+) load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR-mediated SR Ca(2+) leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under β-adrenergic stimulation, Ca(2+) transient amplitude, SR Ca(2+) load, and latency of pro-arrhythmic spontaneous Ca(2+) waves (SCWs) were decreased while RyR-mediated SR Ca(2+) leak was increased in cardiomyocytes from old rabbits. Additionally, with β-adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, which reduced SR Ca(2+) leak, ablated most

  16. Why does the agonist [18F]FP-TZTP bind preferentially to the M2 muscarinic receptor?

    Preferential binding of FP-TZTP at the M2 receptor in vivo led to investigation of [18F]FP-TZTP as a potential PET tracer for Alzheimer's disease, in which a substantial reduction of M2 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 M2 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 Bmax, Ki, and IC50. 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 [18F]FP-TZTP off-rates were determined by measurement of the rate of release into a buffer solution of [18F]FP-TZTP from slide-bound cells that had been preincubated with [18F]FP-TZTP. At equilibrium in vitro, M2 subtype selectivity of [18F]FP-TZTP was not evident. We demonstrated that ATP-dependent mechanisms are not responsible for FP-TZTP M2 selectivity. In vitro off-rate studies from rat brain tissue showed that the off-rate of FP-TZTP varied with the percentage of M2 subtype in the tissue region. The slower dissociation kinetics of FP-TZTP from M2 receptors compared with the four other muscarinic receptor subtypes may be a factor in its M2 selectivity. (orig.)

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

    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.)

  18. CYCLIC CHOLECYSTOKININ ANALOGUES EXHIBIT HIGH BLOOD STABILITY AND BINDING AFFINITY WITH CHOLECYSTOKININ RECEPTOR

    Eun-Ha Joh

    2014-01-01

    Full Text Available Recently, incidence of Cholecystokinin (CCK receptor is recognized as a factor that determines the aggressive phenotype of pancreatic cancer. In this study, a novel Cholecystokinin (CCK analogues; DOTA-Nle-cyclo (Glu-Trp-Met-Asp-Phe-Lys-NH2 (DOTA-cCCK and DOTA-Nle-cyclo (Glu-Trp-Nle-Asp-Phe-Lys-NH2 (DOTA-[Nle]-cCCK were synthesized and radiolabeled and the targeting abilities on the CCK receptor were evaluated for new CCK receptor targeting agents searching. Peptides were prepared through a solid phase synthesis method and their purity was over 98%. DOTA is the chelating agent for 68Ga-labelling, which the peptides were radiolabeled with 68Ga by a high radiolabeling yield (>98%. Peptides were stable over 98% by incubation in mouse blood at 37°C for 2 h. A competitive displacement of 125I-CCK8 on the AR42J human pancreatic carcinoma cells revealed that 50% inhibitory concentration value (IC50 were 12.31 nM of DOTA-cCCK and 1.69 nM of DOTA-[Nle]-cCCK. Stable in the blood of both DOTA-cCCK and DOTA-[Nle]-cCCK, but the binding rate with the CCK receptor on AR42J cells, DOTA-[Nle]-cCCK confirmed better than DOTA-cCCK. Therefore, it is concluded that 68Ga-DOTA-[Nle]-cCCK can be potential candidate as a targeting modality for the CCK receptor over-expressing tumors and further studies to evaluate their biological characteristics are needed.

  19. Metallothionein and a peptide modeled after metallothionein, EmtinB, induce neuronal differentiation and survival through binding to receptors of the low-density lipoprotein receptor family

    Ambjørn, Malene; Asmussen, Johanne W; Lindstam, Mats;

    2008-01-01

    Accumulating evidence suggests that metallothionein (MT)-I and -II promote neuronal survival and regeneration in vivo. The present study investigated the molecular mechanisms underlying the differentiation and survival-promoting effects of MT and a peptide modeled after MT, EmtinB. Both MT and...... granule neurons. By means of surface plasmon resonance MT and EmtinB were found to bind to both megalin and LRP. The bindings were abrogated in the presence of receptor-associated protein-1, an antagonist of the low-density lipoprotein receptor family, which also inhibited MT- and EmtinB-induced neurite...... of cell death (Bim(S)). Finally, evidence is provided that MT and EmtinB activate extracellular signal-regulated kinase, protein kinase B, and cAMP response element binding protein. Altogether, these results strongly suggest that MT and EmtinB induce their neuronal effects through direct binding to...

  20. Elucidating the structures and cooperative binding mechanism of cesium salts to the multitopic ion-pair receptor through density functional theory calculations.

    Sadhu, Biswajit; Sundararajan, Mahesh; Velmurugan, Gunasekaran; Venuvanalingam, Ponnambalam

    2015-09-21

    Designing new and innovative receptors for the selective binding of radionuclides is central to nuclear waste management processes. Recently, a new multi-topic ion-pair receptor was reported which binds a variety of cesium salts. Due to the large size of the receptor, quantum chemical calculations on the full ion-pair receptors are restricted, thus the binding mechanisms are not well understood at the molecular level. We have assessed the binding strengths of various cesium salts to the recently synthesized multi-topic ion-pair receptor molecule using density functional theory based calculations. Our calculations predict that the binding of cesium salts to the receptor predominantly occurs via the cooperative binding mechanism. Cesium and the anion synergistically assist each other to bind favorably inside the receptor. Energy decomposition analysis on the ion-pair complexes shows that the Cs salts are bound to the receptor mainly through electrostatic interactions with small contribution from covalent interactions for large ionic radius anions. Further, QTAIM analysis characterizes the importance of different inter-molecular interactions between the ions and the receptor inside the ion-pair complexes. The role of the crystallographic solvent molecule contributes significantly by ~10 kcal mol(-1) to the overall binding affinities which is quite significant. Further, unlike the recent molecular mechanics (MM) calculations, our calculated binding affinity trends for various Cs ion-pair complexes (CsF, CsCl and CsNO3) are now in excellent agreement with the experimental binding affinity trends. PMID:26227949

  1. Identification of the Calmodulin-Binding Domains of Fas Death Receptor.

    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

  2. MHC class II proteins contain a potential binding site for the verotoxin receptor glycolipid CD77.

    George, T; Boyd, B; Price, M; Lingwood, C; Maloney, M

    2001-11-01

    Globotriaosyl ceramide or CD77 functions as a cell surface receptor for toxins of the Shiga toxin/verotoxin family and as a marker for germinal center stage B-cells. The B-cell protein CD19 and the interferon-alpha receptor possess verotoxin-like amino acid sequences in their extracellular domains, and CD77 has been shown to function in CD19-mediated adhesion and interferon-induced growth inhibition. The Burkitt's lymphoma cell line, Daudi, is similar to germinal center B-cells in their expression of CD77, CD19 and MHC class II molecules. Using the multiple sequence alignment program, ClustalW, we have identified a verotoxin-like amino acid sequence on the beta-chain of human and murine MHC class II molecules. Binding of CD77 at this site could modulate the peptide-binding properties of these MHC class II molecules. Using Western blot analysis of whole cell extracts, we found that CD77-positive Daudi cells have higher levels of HLA-D proteins than VT500 cells, a Daudi-derived CD77-deficient mutant cell line. In contrast, MHC class II-mediated adhesion and surface expression are similar in the two cell lines. Therefore, CD77 could play a functional or regulatory role in MHC class II-mediated functions specifically relating to antigen presentation by B-cells to T helper cells. PMID:11838965

  3. [3H]opipramol labels a novel binding site and sigma receptors in rat brain membranes

    Opipramol (OP), a clinically effective antidepressant with a tricyclic structure, is inactive as an inhibitor of biogenic amine uptake. [3H]Opipramol binds saturably to rat brain membranes (apparent KD = 4 nM, Bmax = 3 pmol/mg of protein). [3H]Opipramol binding can be differentiated into haloperidol-sensitive and -resistant components, with Ki values for haloperidol of 1 nM (Bmax = 1 pmol/mg of protein) and 350 nM (Bmax = 1.9 pmol/mg of protein), respectively. The drug specificity of the haloperidol-sensitive component is the same as that of sigma receptors labeled with (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperdine. The haloperidol-resistant component does not correspond to any known neurotransmitter receptor or uptake recognition site. It displays high affinity for phenothiazines and related structures such as perphenazine, clopenthixol, and flupenthixol, whose potencies are comparable to that of opipramol. Because certain of these drugs are more potent at the haloperidol-resistant opipramol site than in exerting any other action, it is possible that this opipramol-selective site may mediate their therapeutic effects

  4. Reduction of GABA/sub B/ receptor binding induced by climbing fiber degeneration in the rat cerebellum

    When the rat cerebellar climbing fibers degenerated, as induced by lesioning the inferior olive with 3-acetylpyridine (3-AP), GABA/sub B/ receptor binding determined with 3H-(+/-)baclofen was reduced in the cerebellum but not in the cerebral cortex of rats. Computer analysis of saturation data revealed two components of the binding sites, and indicated that decrease of the binding in the cerebellum was due to reduction in receptor density, mainly of the high-affinity sites, the B/sub max/ of which was reduced to one-third that in the control animals. In vitro treatment with 3-AP, of the membranes prepared from either the cerebellum or the cerebral cortex, induced no alteration in the binding sites, thereby indicating that the alteration of GABA/sub B/ sites induced by in vivo treatment with 3-AP is not due to a direct action of 3-AP on the receptor. GABA/sub A/ and benzodiazepine receptor binding labelled with 3H-muscimol and 3H-diazepam, respectively, in both of brain regions was not affected by destruction of the inferior olive. These results provide evidence that some of the GABA/sub B/ sites but neither GABA/sub A/ nor benzodiazepine receptors in the cerebellum are located at the climbing fiber terminals. 28 references, 4 figures, 2 tables

  5. Reduction of GABA/sub B/ receptor binding induced by climbing fiber degeneration in the rat cerebellum

    Kato, K.; Fukuda, H.

    1985-07-22

    When the rat cerebellar climbing fibers degenerated, as induced by lesioning the inferior olive with 3-acetylpyridine (3-AP), GABA/sub B/ receptor binding determined with /sup 3/H-(+/-)baclofen was reduced in the cerebellum but not in the cerebral cortex of rats. Computer analysis of saturation data revealed two components of the binding sites, and indicated that decrease of the binding in the cerebellum was due to reduction in receptor density, mainly of the high-affinity sites, the B/sub max/ of which was reduced to one-third that in the control animals. In vitro treatment with 3-AP, of the membranes prepared from either the cerebellum or the cerebral cortex, induced no alteration in the binding sites, thereby indicating that the alteration of GABA/sub B/ sites induced by in vivo treatment with 3-AP is not due to a direct action of 3-AP on the receptor. GABA/sub A/ and benzodiazepine receptor binding labelled with /sup 3/H-muscimol and /sup 3/H-diazepam, respectively, in both of brain regions was not affected by destruction of the inferior olive. These results provide evidence that some of the GABA/sub B/ sites but neither GABA/sub A/ nor benzodiazepine receptors in the cerebellum are located at the climbing fiber terminals. 28 references, 4 figures, 2 tables.

  6. PET evaluation of the relationship between D2 receptor binding and glucose metabolism in patients with parkinsonism

    The objective of this study was to clarify the relationship between D2 receptor binding and the cerebral metabolic rate for glucose (CMRGlu) in patients with parkinsonism, we simultaneously measured both of these factors, and then compared the results. The subjects consisted of 24 patients: 9 with Parkinson's disease (PD), 3 with Juvenile Parkinson's disease (JPD), 9 with multiple system atrophy (MSA), and 3 with progressive supranuclear palsy (PSP). The striatal D2 receptor binding was measured by the C-11 raclopride transient equilibrium method. CMRGlu was investigated by the F-18 fluorodeoxyglucose autoradiographic method. The D2 receptor binding in both the caudate nucleus and putamen showed a positive correlation with the CMRGlu in the PD-JPD group, but the two parameters demonstrated no correlation in the MSA-PSP group. The left/right (L/R) ratio of D2 receptor binding in the putamen showed a positive correlation with that of CMRGlu in the MSA-PSP group, while the two demonstrated no correlation in the PD-JPD group. Our PET study showed striatal D2 receptor binding and the CMRGlu to be closely related in patients with parkinsonism, even though the results obtained using the L/R ratios tended to differ substantially from those obtained using absolute values. The reason for this difference is not clear, but this finding may reflect the pathophysiology of these disease entities. (author)

  7. The structure of the follistatin:activin complex reveals antagonism of both type I and type II receptor binding

    Thompson, T.B.; Lerch, T.F.; Cook, R.W.; Woodruff, T.K.; Jardetzky, T.S. (NWU)

    2010-03-08

    TGF-{beta} ligands stimulate diverse cellular differentiation and growth responses by signaling through type I and II receptors. Ligand antagonists, such as follistatin, block signaling and are essential regulators of physiological responses. Here we report the structure of activin A, a TGF-{beta} ligand, bound to the high-affinity antagonist follistatin. Two follistatin molecules encircle activin, neutralizing the ligand by burying one-third of its residues and its receptor binding sites. Previous studies have suggested that type I receptor binding would not be blocked by follistatin, but the crystal structure reveals that the follistatin N-terminal domain has an unexpected fold that mimics a universal type I receptor motif and occupies this receptor binding site. The formation of follistatin:BMP:type I receptor complexes can be explained by the stoichiometric and geometric arrangement of the activin:follistatin complex. The mode of ligand binding by follistatin has important implications for its ability to neutralize homo- and heterodimeric ligands of this growth factor family.

  8. Insights into the binding of Phenyltiocarbamide (PTC agonist to its target human TAS2R38 bitter receptor.

    Xevi Biarnés

    Full Text Available Humans' bitter taste perception is mediated by the hTAS2R subfamily of the G protein-coupled membrane receptors (GPCRs. Structural information on these receptors is currently limited. Here we identify residues involved in the binding of phenylthiocarbamide (PTC and in receptor activation in one of the most widely studied hTAS2Rs (hTAS2R38 by means of structural bioinformatics and molecular docking. The predictions are validated by site-directed mutagenesis experiments that involve specific residues located in the putative binding site and trans-membrane (TM helices 6 and 7 putatively involved in receptor activation. Based on our measurements, we suggest that (i residue N103 participates actively in PTC binding, in line with previous computational studies. (ii W99, M100 and S259 contribute to define the size and shape of the binding cavity. (iii W99 and M100, along with F255 and V296, play a key role for receptor activation, providing insights on bitter taste receptor activation not emerging from the previously reported computational models.

  9. Reconstitution of rate brain μ opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go

    Reconstitution of purified μ opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. The purified μ opioid receptor (pI 5.6) migrated as a single Mr 58,000 polypeptide by NaDodSO4/PAGE, a value identical to that obtained by affinity cross-linking purified μ receptors. When purified μ receptors were reconstituted with purified Gi, the G protein that mediates the inhibition of adenylate cyclase, the displacement of [3H]naloxone binding by [D-Ala2,MePhe4,Gly-ol5]enkephalin was increased 215-fold; this increase was abolished by adding 100 μM guanosine 5'-[γ-thio]triphosphate. Similar increases in agonist displacement of [3H]naloxone binding (33-fold) and its abolition by guanosine 5'-[γ-thio]triphosphate were observed with Go, the G protein of unknown function, but not with the v-Ki-ras protein p.21. The stoichiometry was such that the stimulation of 1 mol of μ receptor led to the binding of [3H]guanosine 5'-[β,γ-imido]triphosphate to 2.5 mol of Gi or to 1.37 mol of Go. These results suggest that the purified μ opioid receptor is functionally coupled to Gi and Go in the reconstituted phospholipid vesicles

  10. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

    Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map the distribution on frozen skin sections of an extracellular epitope on the EGF receptor. The [125I]EGF binding experiments showed accessible, unoccupied EGF receptors to be present on the epidermal basal cells (with reduced binding to spinous cells), the basal cells of the hair shaft and sebaceous gland, the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing presumptive cortex cells, excluding the medulla, lying around and above the upper dermal papilla of anagen hair follicles, epithelial cells around the lower dermal papilla region, and in some tissue samples the cell margins of the viable differentiating layers of the epidermis. In a control study, to clarify whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar, indicating that, at least for SVK14 cells, EGF-R1 binding provides a reliable marker for EGF binding. Explanations for the discrepancies between these two methods for determining EGF receptor distribution in human skin are discussed

  11. Binding of receptor-recognized forms of alpha2-macroglobulin to the alpha2-macroglobulin signaling receptor activates phosphatidylinositol 3-kinase.

    Misra, U K; Pizzo, S V

    1998-05-29

    Ligation of the alpha2-macroglobulin (alpha2M) signaling receptor by receptor-recognized forms of alpha2M (alpha2M*) initiates mitogenesis secondary to increased intracellular Ca2+. We report here that ligation of the alpha2M signaling receptor also causes a 1. 5-2.5-fold increase in wortmannin-sensitive phosphatidylinositol 3-kinase (PI3K) activity as measured by the quantitation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 formation was alpha2M* concentration-dependent with a maximal response at approximately 50 pM ligand concentration. The peak formation of PIP3 occurred at 10 min of incubation. The alpha2M receptor binding fragment mutant K1370R which binds to the alpha2M signaling receptor activating the signaling cascade, increased PIP3 formation by 2-fold. The mutant K1374A, which binds very poorly to the alpha2M signaling receptor, did not cause any increase in PIP3 formation. alpha2M*-induced DNA synthesis was inhibited by wortmannin. 1, 2Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethylester a chelator of intracellular Ca2+, drastically reduced alpha2M*-induced increases in PIP3 formation. We conclude that PI3K is involved in alpha2M*-induced mitogenesis in macrophages and intracellular Ca2+ plays a role in PI3K activation. PMID:9593670

  12. Changes in dynamical behavior of the retinoid X receptor DNA-binding domain upon binding to a 14 base-pair DNA half site.

    van Tilborg, P J; Czisch, M; Mulder, F A; Folkers, G E; Bonvin, A M; Nair, M; Boelens, R; Kaptein, R

    2000-08-01

    The retinoid X receptor (RXR) is a prominent member of the nuclear receptor family of ligand-inducible transcription factors. Many proteins of this family exert their function as heterodimers with RXR as a common upstream partner. Studies of the DNA-binding domains of several nuclear receptors reveal differences in structure and dynamics, both between the different proteins and between the free- and DNA-bound receptor DBDs. We investigated the differences in dynamics between RXR free in solution and in complex with a 14 base-pair oligonucleotide, using (1)H and (15)N relaxation studies. Nano- to picosecond dynamics were probed on (15)N, employing Lipari-Szabo analysis with an axially symmetric tumbling model to estimate the exchange contributions to the transverse relaxation rates. Furthermore, milli- to microsecond dynamics were estimated qualitatively for (1)H and (15)N, using CPMG-HSQC and CPMG-T(2) measurements with differential pulse spacing. RXR shows hardly any nano- to picosecond time-scale internal motion. Upon DNA binding, the order parameters show a tiny increase. Dynamics in the milli- to microsecond time scale is more prevalent. It is localized in the first and second zinc fingers of the free RXR. Upon DNA-binding, exchange associated with specific/aspecific DNA-binding of RXR is observed throughout the sequence, whereas conformational flexibility of the D-box and the second zinc finger of RXR is greatly reduced. Since this DNA-binding induced folding transition occurs remote from the DNA in a region which is involved in protein-protein interactions, it may very well be related to the cooperativity of dimeric DNA binding. PMID:10913286

  13. Ability of luteinizing hormone releasing hormone-Pseudomonas aeruginosa exotoxin 40 binding to LHRH receptor on human liver cancer cells

    Shou-Liang Gong; Gang Zhao; Hong-Guang Zhao; Wen-Tian Lü; Guang-Wei Liu; Ping Zhu

    2004-01-01

    AIM: To explore the ability of recombinant toxin luteinizing hormone releasing hormone-Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40) anH binding to LHRH receptor(LHRHR) on the membrane surfa ogf hman liver cancer HEPG cells.METHODS: LHRH was beled by using 125I with enzymatic reaction. The affinity and receptor volume of LHRH-PE40and LHRH binding to LHRHR on the membrane surface of human liver cancer cells were measured with radioligand receptor assay.RESULTS:The specific activity of LHRH labeled with 125I was 2.7×104 kBq/μL, and its radiochemical purity reached to 99.2-99.7%. The binding of 125I to LHRH was maximal for 240 min in the warm cultivation, and this binding was stabilized. The inhibiting rates of 125I-LHRH and LHRH on the proliferation of human liver cancer HEPG cells were not significantly different. On the basis of the saturation curve of 125I-LHRH binding to the membrane LHRHR of HEPG cells, 125I-LHRH of 1×105 cpm was selected for radioligand receptor assay. The affinity constants (Kd) of LHRH-PE40and LHRH bively,and their receptor volumes were 0.37±0.15 μmol/g and0.42±0.13 μmol/g, respectively. The binding of LHRH-PE40to the membrane proteinof normal liver cells was not observed.CONCLUSION: The recombinant toxin LHRH-PE40 binding to the membrane surface of LHRHR of human liver cancer HEPG cells was very strong, while the specific binding of it to normal liver cells was not observed. The results provide an important experimental basis for the clinical application of LHRH-PE.

  14. Correlation of pharmacological activity and receptor binding of guanabenz during development

    Many studies to elucidate the pharmacokinetic, pharmacodynamic, and molecular pharmacological profile of guanabenz, an α2-adrenergic receptor agonist, have been reported; however, the effects of this drug on the developing fetus have been largely ignored. The ability of a drug to alter fetal cardiovascular activity is dependent upon both the penetration of the drug across the placenta and upon maturation of the system(s) through which the drug exerts its effects. Consequently, it is hypothesized that the pharmacological activity of guanabenz on the fetus is influenced both by placenta actions on drug transfer and the time course of development of the central and/or peripheral α2adrenergic receptor system(s) through which the drug exerts its effects. Guanabenz (GB) when administered to the maternal sheep elicited a cardiovascular response similar to that observed in rats, dogs, and humans. An initial, transient increase in mean arterial pressure (MAP) was followed by a sustained decrease in MAP. This decrease in MAP was accompanied by a prolonged decrease in heart rate (HR). No cardiovascular response to maternally administered GB was observed in the fetal lamb. Pharmacokinetic studies revealed that GB is widely distributed in the pregnant ewe; however, the placenta appears to act as a relative barrier to the transfer of GB into the fetal compartment. In order to ascertain if the mature fetal lamb had developed functional α2-adrenergic receptors, GB was administered directly to the fetus. A transient increase in MAP was elicited; however, no prolonged decrease in either MAP or HR occurred. Following validation of [3H]guanabenz ([3H]GB) binding assay in rat cerebral cortex, studies to correlate [3H]GB binding in sheep cortex with pharmacodynamic response was conducted

  15. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

    Whittle, James R.R.; Zhang, Ruijun; Khurana, Surender; King, Lisa R.; Manischewitz, Jody; Golding, Hana; Dormitzer, Philip R.; Haynes, Barton F.; Walter, Emmanuel B.; Moody, M. Anthony; Kepler, Thomas B.; Liao, Hua-Xin; Harrison, Stephen C. (Harvard-Med); (Novartis); (US-FDA); (Duke)

    2011-09-20

    Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.

  16. Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.

    Fotticchia, Iolanda; Guarnieri, Daniela; Fotticchia, Teresa; Falanga, Andrea Patrizia; Vecchione, Raffaele; Giancola, Concetta; Netti, Paolo Antonio

    2016-08-01

    Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier. PMID:27100851

  17. The Receptor-Binding Domain in the VP1u Region of Parvovirus B19.

    Leisi, Remo; Di Tommaso, Chiarina; Kempf, Christoph; Ros, Carlos

    2016-03-01

    Parvovirus B19 (B19V) is known as the human pathogen causing the mild childhood disease erythema infectiosum. B19V shows an extraordinary narrow tissue tropism for erythroid progenitor cells in the bone marrow, which is determined by a highly restricted uptake. We have previously shown that the specific internalization is mediated by the interaction of the viral protein 1 unique region (VP1u) with a yet unknown cellular receptor. To locate the receptor-binding domain (RBD) within the VP1u, we analyzed the effect of truncations and mutations on the internalization capacity of the recombinant protein into UT7/Epo cells. Here we report that the N-terminal amino acids 5-80 of the VP1u are necessary and sufficient for cellular binding and internalization; thus, this N-terminal region represents the RBD required for B19V uptake. Using site-directed mutagenesis, we further identified a cluster of important amino acids playing a critical role in VP1u internalization. In silico predictions and experimental results suggest that the RBD is structured as a rigid fold of three α-helices. Finally, we found that dimerization of the VP1u leads to a considerably enhanced cellular binding and internalization. Taken together, we identified the RBD that mediates B19V uptake and mapped functional and structural motifs within this sequence. The findings reveal insights into the uptake process of B19V, which contribute to understand the pathogenesis of the infection and the neutralization of the virus by the immune system. PMID:26927158

  18. Genome-wide binding and transcriptome analysis of human farnesoid X receptor in primary human hepatocytes.

    Le Zhan

    Full Text Available Farnesoid X receptor (FXR, NR1H4 is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing (ChIP-seq, with results indicating that FXR may be involved in regulating diverse pathways in liver. However, limited information exists for the functions of human FXR and the suitability of using murine models to study human FXR functions.In the current study, we performed ChIP-seq in primary human hepatocytes (PHHs treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq and RNA microarray were performed for GW4064 or control treated PHHs and wild type mouse livers, respectively.ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment.In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions.

  19. Expression cloning of a cDNA encoding the murine interleukin 4 receptor based on ligand binding

    Harada, N.; Castle, B.E.; Gorman, D.M.; Itoh, A.; Schreurs, J.; Barrett, R.L.; Howard, M.; Miyajima, A. (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA (USA))

    1990-02-01

    Interleukin 4 (IL-4) is a potent mediator of growth and differentiation for various lymphoid and myeloid cells. To isolate a cDNA encoding the murine IL-4 receptor, the authors have developed an expression cloning method that uses biotinylated ligand as a probe and that may be generally applicable to cloning of receptor genes. COS-7 cells transiently transfected with the cloned full-length cDNA bind murine IL-4 specifically with a K{sub d} = 165 pM. Crosslinking of {sup 125}I-labeled IL-4 to COS-7 cells transfected with the cDNA reveals binding to proteins of 120-140 kDa. IL-4-responsive cells also express IL-4-binding proteins of 120-140 kDa but show additional bands at 60-70 kDa; the relationship of the smaller proteins to the larger ones is unclear. The nucleotide sequence indicates that the full-length cDNA encodes 810 amino acids including the signal sequence. While no consensus sequence for protein kinases is present in the cytoplasmic domain, a sequence comparison with the erythropoietin receptor, the IL-6 receptor, and the {beta} chain of the IL-2 receptor reveals a significant homology in the extracellular domain, indicating that the IL-4 receptor is a member of a cytokine receptor family.

  20. Binding characteristics of brain-derived neurotrophic factor to its receptors on neurons from the chick embryo

    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

  1. Neonicotinoid binding, toxicity and expression of nicotinic acetylcholine receptor subunits in the aphid Acyrthosiphon pisum.

    Emiliane Taillebois

    Full Text Available Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI, thiamethoxam (TMX and clothianidin (CLT. Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16 ± 0.04 nM and 41.7 ± 5.9 nM and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008 ± 0.002 nM and 1.135 ± 0.213 nM. Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml and TMX (LC50 = 0.034 µg/ml were more toxic than CLT (LC50 = 0.118 µg/ml. The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.

  2. Membrane binding mode of intrinsically disordered cytoplasmic domains of T cell receptor signaling subunits depends on lipid composition

    Intrinsically disordered cytoplasmic domains of T cell receptor (TCR) signaling subunits including ζcyt and CD3εcyt all contain one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor triggering. Membrane binding-induced helical folding of ζcyt and CD3εcyt ITAMs is thought to control TCR activation. However, the question whether or not lipid binding of ζcyt and CD3εcyt is necessarily accompanied by a folding transition of ITAMs remains open. In this study, we investigate whether the membrane binding mechanisms of ζcyt and CD3εcyt depend on the membrane model used. Circular dichroic and fluorescence data indicate that binding of ζcyt and CD3εcyt to detergent micelles and unstable vesicles is accompanied by a disorder-to-order transition, whereas upon binding to stable vesicles these proteins remain unfolded. Using electron microscopy and dynamic light scattering, we show that upon protein binding, unstable vesicles fuse and rupture. In contrast, stable vesicles remain intact under these conditions. This suggests different membrane binding modes for ζcyt and CD3εcyt depending on the bilayer stability: (1) coupled binding and folding, and (2) binding without folding. These findings explain the long-standing puzzle in the literature and highlight the importance of the choice of an appropriate membrane model for protein-lipid interactions studies.

  3. Computational Estimates of Binding Affinities for Estrogen Receptor Isoforms in Rainbow Trout

    Shyu, Conrad

    2009-01-01

    Molecular dynamics simulations are performed to determine the binding affinities between the hormone 17 beta-estradiol (E2) and different estrogen receptor (ER) isoforms in the rainbow trout (Oncorhynchus mykiss). Previous studies have demonstrated that a recent, unique gene duplication of the ER alpha subtype created two isoforms ER alpha 1 and ER alpha 2, and an early secondary split of ER beta produced two distinct isoforms of ER beta 1 and ER beta 2 based on the phylogenetic analysis. The objective of our computational studies is to provide insight into the underlying evolutionary selection pressure on the ER isoforms. Our results show that E2 binds preferentially to ER alpha 1. This finding corresponds to the experimental results as the ERs evolved from gene duplication events are frequently free from selective pressure and should exhibit no deleterious effects. The E2, however, only binds slightly better to ER beta 2. Both isoforms remain competitive. This finding reflects the fact that since ER beta 2 ...

  4. Adenosine A2A receptor binding profile of two antagonists, ST1535 and KW6002: consideration on the presence of atypical adenosine A2A binding sites

    Teresa Riccioni

    2010-08-01

    Full Text Available Adenosine A2A receptors seem to exist in typical (more in striatum and atypical (more in hippocampus and cortex subtypes. In the present study, we investigated the affinity of two adenosine A2A receptor antagonists, ST1535 [2 butyl -9-methyl-8-(2H-1,2,3-triazol 2-yl-9H-purin-6-xylamine] and KW6002 [(E-1,3-diethyl-8-(3,4-dimethoxystyryl-7-methyl-3,7-dihydro-1H-purine-2,6,dione] to the “typical” and “atypical” A2A binding sites. Affinity was determined by radioligand competition experiments in membranes from rat striatum and hippocampus. Displacement of the adenosine analog [3H]CGS21680 [2-p-(2-carboxyethylphenethyl-amino-5’-N-ethylcarbox-amidoadenosine] was evaluated in the absence or in the presence of either CSC [8-(3-chlorostyryl-caffeine], an adenosine A2A antagonist that pharmacologically isolates atypical binding sites, or DPCPX (8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor antagonist that pharmacologically isolates typical binding site. ZM241385 [84-(2-[7-amino-2-(2-furyl [1,2,4]-triazol[2,3-a][1,3,5]triazin-5-yl amino]ethyl phenol] and SCH58261 [(5-amino-7-(β-phenylethyl-2-(8-furylpyrazolo(4,3-e-1,2,4-triazolo(1,5-c pyrimidine], two other adenosine A2A receptor antagonists, which were reported to differently bind to atypical and typical A2A receptors, were used as reference compounds. ST1535, KW6002, ZM241385 and SCH58261 displaced [3H]CGS21680 with higher affinity in striatum than in hippocampus. In hippocampus, no typical adenosine A2A binding was detected, and ST1535 was the only compound that occupied atypical A2A adenosine receptors. Present data are explained in terms of heteromeric association among adenosine A2A, A2B and A1 receptors, rather than with the presence of atypical A2A receptor subtype.

  5. Distinction between high-affinity [3H]phencyclidine binding sites and muscarinic receptors in guinea-pig ileum muscle

    [3H]Phencyclidine ([3H]PCP) binding was studied in guinea-pig ileum muscle membranes. Specific binding of [3H]PCP was time dependent, reversible and saturable, with an equilibrium dissociation constant of 154 nM and maximum binding of 12.9 pmol/mg of protein at pH 9. Its pH dependency suggests that the un-ionized PCP is the pharmacologically active form. The binding site was on a protein which was sensitive to heat, proteolytic enzymes and the carboxylic group reagent dicyclohexylcarbodiimide, but insensitive to phospholipase A and C, concanavalin A, dithiothreitol and N-ethylmaleimide. Specific [3H]PCP binding was displaced effectively by several PCP analogs and Ca++ channel antagonists including verapamil, to which these sites had a high affinity. These high-affinity PCP-binding sites were found at a much higher concentration in the same membrane preparation than muscarinic receptor sites identified by their specific binding of [3H]quinuclidinyl benzilate. PCP bound to both sites, but with a lower affinity to the muscarinic receptor sites. The PCP and muscarinic receptor sites differed in their sensitivities to pH and drug specifities

  6. Identification of residues on human receptor DPP4 critical for MERS-CoV binding and entry

    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

  7. Identification of residues on human receptor DPP4 critical for MERS-CoV binding and entry

    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.

  8. Proliferative responses and binding properties of hematopoietic cells transfected with low-affinity receptors for leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor.

    Gearing, D P; Ziegler, S F; Comeau, M R; Friend, D; Thoma, B; Cosman, D; Park, L.; Mosley, B

    1994-01-01

    Specific low-affinity receptors for leukemia inhibitory factor (LIF), oncostatin M (OSM; gp130), and ciliary neurotrophic factor (CNTF; receptor alpha, CNTFR alpha) may be utilized in various combinations to generate high-affinity binding sites and signal transduction. We have tested the ability of combinations of these receptors to transduce a proliferative signal in BAF-B03 cells. Coexpression of the LIF receptor and gp130 in these cells conferred high-affinity LIF and OSM binding and respo...

  9. Contrasting roles of leu(356) in the human CCK(1) receptor for antagonist SR 27897 and agonist SR 146131 binding.

    Gouldson, P; Legoux, P; Carillon, C; Delpech, B; Le Fur, G; Ferrara, P; Shire, D

    1999-11-01

    A new highly specific, potent non-peptide agonist for the cholecystokinin subtype 1 receptor (CCK(1)), SR 146131 (2-[4-(4-chloro-2, 5-dimethoxyphenyl)-5-(2-cyclohexyl-ethyl)-thiazol-2-ylcarbamoyl ]-5, 7-dimethyl-indol-1-yl-1-acetic acid) was recently described [Bignon, E., Bachy, A., Boigegrain, R., Brodin, R., Cottineau, M., Gully, D., Herbert, J.-M., Keane, P., Labie, C., Molimard, J.-C., Olliero, D., Oury-Donat, F., Petereau, C., Prabonneaud, V., Rockstroh, M.-P., Schaeffer, P., Servant, O.Thurneyssen, O., Soubrié, P., Pascal, M., Maffrand, J.-P., Le Fur, G., 1999. SR 146131: a new, potent, orally active and selective non-peptide cholecystokinin subtype I receptor agonist: I. In vitro studies. J. Pharmacol. Exp. Ther. 289, 742-751]. From binding and activity assays with chimeric constructs of human CCK(1) and the cholecystokinin subtype 2 receptor (CCK(2)) and receptors carrying point mutations, we show that Leu(356), situated in transmembrane domain seven in the CCK(1) receptor, is a putative contact point for SR 146131. In contrast, Leu(356) is probably not in contact with the CCK(1) receptor specific antagonist SR 27897 (1-[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl indoyl]acetic acid), a compound structurally related to SR 146131, since its replacement by alanine, histidine or asparagine gave receptors having wild-type CCK(1) receptor SR 27897 binding affinity. Previous mutational analysis of His(381), the cognate position in the rat CCK(2) receptor, had implicated it as being involved in subtype specificity for SR 27897, results which we confirm with corresponding mutations in the human CCK(2) receptor. Moreover, binding and activity assays with the natural CCK receptor agonist, CCK-8S, show that CCK-8S is more susceptible to the mutations in that position in the CCK(1) receptor than in the CCK(2) receptor. The results suggest different binding modes for SR 27897, SR 146131 and CCK-8S in each CCK receptor subtype. PMID:10594328

  10. Specific binding and laterality of human extrastriatal dopamine D2/D3 receptors in late onset type 1 alcoholic patients.

    Kuikka, J T; Repo, E; Bergström, K A; Tupala, E; Tiihonen, J

    2000-09-29

    Late onset type 1 alcoholism has been suggested to be associated with decreased dopaminergic transmission. Our hypothesis was that late onset type 1 alcoholics have also abnormal extrastriatal dopamine D(2)/D(3) receptor distribution. We performed binding, heterogeneity and laterality analysis of extrastriatal and striatal dopamine D(2)/D(3) receptors in nine late onset male alcoholics and in 12 age-matched healthy males. A radioligand, [(123)I]epidepride was used in high resolution single-photon emission tomography (SPET). Specific binding of epidepride in the left temporal pole was significantly (Pepidepride distribution observed in control males (0.89+/-0.19 vs. 1.10+/-0.19; P<0.05). The results suggest that the specific binding of dopamine D(2)/D(3) receptors in late type 1 alcoholics is decreased and its laterality in the temporal brain is altered from normal. PMID:10996449

  11. Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

    Antoine, Thomas; Ott, David; Ebell, Katharina; Hansen, Kerrin; Henry, Luc; Becker, Frank; Hannus, Stefan

    2016-06-01

    G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs. PMID:26954998

  12. Homology-modeled ligand-binding domains of medaka estrogen receptors and androgen receptors: A model system for the study of reproduction

    Estrogen and androgen and their receptors play critical roles in physiological processes such as sexual differentiation and development. Using the available structural models for the human estrogen receptors alpha and beta and androgen receptor as templates, we designed in silico agonist and antagonist models of medaka estrogen receptor (meER) alpha, beta-1, and beta-2, and androgen receptor (meAR) alpha and beta. Using these models, we studied (1) the structural relationship between the ligand-binding domains (LBDs) of ERs and ARs of human and medaka, and (2) whether medaka ER and AR can be potential models for studying the ligand-binding activities of various agonists and antagonists of these receptors by docking analysis. A high level of conservation was observed between the sequences of the ligand-binding domains of meERα and huERα, meERβ1 and huERβ, meERβ2, and huERβ with 62.8%, 66.4%, and 65.1% identity, respectively. The sequence conservation between meARα and huAR, meARβ, and huAR was found with 70.1% and 61.0% of identity, respectively. Thirty-three selected endocrine disrupting chemicals (EDCs), including both agonists and antagonists, were docked into the LBD of ER and AR, and the corresponding docking score for medaka models and human templates were calculated. In order to confirm the conservation of the overall geometry and the binding pocket, the backbone root mean square deviation (RMSD) for Cα atoms was derived from the structure superposition of all 10 medaka homology models to the six human templates. Our results suggested conformational conservation between the ERs and ARs of medaka and human, Thus, medaka could be highly useful as a model system for studies involving estrogen and androgen interaction with their receptors.

  13. Construction of a high affinity zinc binding site in the metabotropic glutamate receptor mGluR1

    Jensen, Anders A.; Sheppard, P O; Jensen, L B;

    2001-01-01

    The metabotropic glutamate receptors (mGluRs) belong to family C of the G-protein-coupled receptor (GPCR) superfamily. The receptors are characterized by having unusually long amino-terminal domains (ATDs), to which agonist binding has been shown to take place. Previously, we have constructed a...... "closed" conformation, and thus stabilizing a more or less inactive "open" form of the ATD. This study presents the first metal ion site constructed in a family C GPCR. Furthermore, it is the first time a metal ion site has been created in a region outside of the seven transmembrane regions of a GPCR and...... the loops connecting these. The findings offer valuable insight into the mechanism of ATD closure and family C receptor activation. Furthermore, the findings demonstrate that ATD regions other than those participating in agonist binding could be potential targets for new generations of ligands for...

  14. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl2) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl2 and MeHg on [3H]-quinuclidinyl benzilate ([3H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B max) and ligand affinity (K d). Subsequently, samples were exposed to HgCl2 or MeHg to derive IC50 values and inhibition constants (K i). Results demonstrate that HgCl2 is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [3H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies

  15. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    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

  16. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    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.

  17. Monomer-dimer model explains the results of radiation inactivation: binding characteristics of insulin receptor purified from human placenta

    The technique of radiation inactivation has been used on highly purified human placental insulin receptor in order to determine the functional molecular size responsible for the insulin binding and to evaluate the affinity regulator hypothesis, which has been proposed to explain the increase in specific insulin binding to rat liver membranes observed at low radiation does. Three different types of inactivation curves were observed: (1) biphasic with an enhanced binding activity after exposure to low radiation doses, (2) nonlinear with no change in binding activity after exposure to low radiation doses, and (3) linear with a loss in the binding activity with increasing radiation exposures. A monomer-dimer model was the simplest model that best described the three types of radiation inactivation curves observed. The model predicts that an increase in insulin binding activity would result after exposure to low radiation doses when the initial dimer/monomer ratio is equal to or greater than 1 and a monomer is more active than a dimer. The monomer size of the binding activity was estimated to be 227,000 daltons by this model. To substantiate this model, the purified receptor was fractionated by Sepharose CL-6B chromatography. The insulin binding profile of this column indicated two peaks. These studies suggest that the affinity regulator does not exist as a separate structural protein but is due to the dimeric form of the receptor. The dimeric form (α2β2) possesses a much lower specific activity for insulin binding than does the monomeric αβ form (under the standard conditions), but the dimeric structure is necessary to observe the negative cooperative binding isotherm

  18. Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B

    Stofega, M R; Herrington, J; Billestrup, Nils;

    2000-01-01

    Binding of GH to GH receptor (GHR) rapidly and transiently activates multiple signal transduction pathways that contribute to the growth-promoting and metabolic effects of GH. While the events that initiate GH signal transduction, such as activation of the Janus tyrosine kinase JAK2, are beginnin...

  19. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains

    Behrendt, N; Ronne, E; Dano, K

    1996-01-01

    . This result shows that in addition to D1, which has an established function in ligand binding (Behrendt, N., Ploug, M., Patthy, L., Houen, G., Blasi, F., and Dano, K. (1991) J. Biol. Chem. 266, 7842-7847), D3 has an important role in governing a high affinity in the intact receptor. Real-time biomolecular...

  20. Structural Changes in the Lectin Domain of CD23, the Low-Affinity IgE Receptor, upon Calcium Binding

    Wurzburg, Beth A.; Tarchevskaya, Svetlana S.; Jardetzky, Theodore S. (NWU)

    2010-03-08

    CD23, the low-affinity receptor for IgE (Fc{var_epsilon}RII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca{sup 2+}. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in human CD23. Conformational differences between the apo and Ca{sup 2+} bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.

  1. Autoradiographic comparison of [125I]epidepride and [125I]NCQ 298 binding to human brain extrastriated dopamine receptors.

    Hall, H; Halldin, C; Jerning, E; Osterlund, M; Farde, L; Sedvall, G

    1997-07-01

    Extrastriatal D2-dopamine receptors can be visualised in the monkey and human brain using the benzamides [11C]- and [76Br]FLB 457 in PET and [123I]epidepride in SPECT but not with the salicylamide analogues [76Br]FLB 463 and [123I]NCQ 298. To clarify the background for the differences in binding seen in vivo, we have compared the in vitro binding of [125I]epidepride and [123I]NCQ 298, using human whole hemisphere autoradiography. The images obtained with any radioligand showed detailed distribution with very dense binding in the putamen and the caudate nucleus and with the same detailed extrastriatal distribution. Thus, the divergent results obtained in vivo cannot be explained by different binding properties of the extrastriatal receptors. PMID:9290072

  2. Autoradiographic comparison of [125I]epidepride and [125I]NCQ 298 binding to human brain extrastriated dopamine receptors

    Extrastriatal D2-dopamine receptors can be visualized in the monkey and human brain using the benzamides [11C]- and [76Br]FLB 457 in PET and [123I]epidepride in SPECT but not with the salicylamide analogues [76Br]FLB 463 and [123I]NCQ 298. To clarify the background for the differences in binding seen in vivo, we have compared the in vitro binding of [125I]epidepride and [125I]NCQ 298, using human whole hemisphere autoradiography. The images obtained with any radioligand showed detailed distribution with very dense binding in the putamen and the caudate nucleus and with the same detailed extrastriatal distribution. Thus, the divergent results obtained in vivo cannot be explained by different binding properties of the extrastriatal receptors

  3. Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation

    Benjamin J Tillotson; Goulatis, Loukas I.; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V.

    2015-01-01

    The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a s...

  4. Multiple Transmembrane Binding Sites for p-Trifluoromethyldiazirinyl-etomidate, a Photoreactive Torpedo Nicotinic Acetylcholine Receptor Allosteric Inhibitor*

    Hamouda, Ayman K.; Stewart, Deirdre S.; Husain, S. Shaukat; Cohen, Jonathan B.

    2011-01-01

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [3H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[3H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our unders...

  5. Kinetics of Carbamylcholine Binding to Membrane-Bound Acetylcholine Receptor Monitored by Fluorescence Changes of a Covalently Bound Probe

    Dunn, Susan M.J.; Blanchard, Steven G.; Raftery, Michael A.

    1980-01-01

    The fluorescent probe 5-(iodoacetamido)salicylic acid has been used to alkylate acetylcholine receptor enriched membrane fragments from Torpedo californica following their reduction with low concentrations of dithiothreitol. This modification did not affect the equilibrium binding of carbamylcholine to the receptor. The fluorescence of bound 5-(iodoacetamido)salicylic acid was enhanced when the labeled membrane fragments were mixed with carbamylcholine. This increase in fluorescence was ab...

  6. Extended and Structurally Supported Insights into Extracellular Hormone Binding, Signal Transduction and Organization of the Thyrotropin Receptor

    Krause, Gerd; Kreuchwig, Annika; Kleinau, Gunnar

    2012-01-01

    The hormone thyrotropin (TSH) and its receptor (TSHR) are crucial for the growth and function of the thyroid gland. The TSHR is evolutionary linked with the receptors of follitropin (FSHR) and lutropin/choriogonadotropin (LHR) and their sequences and structures are similar. The extracellular region of TSHR contains more than 350 amino acids and binds hormone and antibodies. Several important questions related to functions and mechanisms of TSHR are still not comprehensively understood. One ma...

  7. High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin

    Saris, Jasper; Derkx, Frans; Bruin, René; Dekkers, Dick; Lamers, Jos; Saxena, Pramod Ranjan; Schalekamp, Maarten; Danser, Jan

    2001-01-01

    textabstractMannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell ...

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

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

    1991-01-01

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

  9. Elimination of Fc receptor binding of human immunoglobulin G in immunofluorescence assays for herpes simplex virus antibodies.

    Gallo, D

    1986-01-01

    The binding of human immunoglobulin G by Fc receptors in herpes simplex virus (HSV)-infected cells can cause false-positive interpretations in the immunofluorescence test for HSV antibody. When the infected cell smears were treated with 10% glacial acetic acid for 5 min and rinsed in phosphate-buffered saline before the immunofluorescence test was performed, the Fc receptors were completely inactivated, resulting in a reliable method for HSV antibody detection.

  10. Probing the structure and function of the estrogen receptor ligand binding domain by analysis of mutants with altered transactivation characteristics.

    Eng, F C; Lee, H.S.; Ferrara, J; Willson, T M; White, J H

    1997-01-01

    We have developed a genetic screen for the yeast Saccharomyces cerevisiae to isolate estrogen receptor (ER) mutants with altered transactivation characteristics. Use of a "reverse" ER, in which the mutagenized ligand binding domain was placed at the N terminus of the receptor, eliminated the isolation of truncated constitutively active mutants. A library was screened with a low-affinity estrogen, 2-methoxyestrone (2ME), at concentrations 50-fold lower than those required for activation of the...

  11. Design and synthesis of a stable oxidized phospholipid mimic with specific binding recognition for macrophage scavenger receptors

    Turner, William W; Hartvigsen, Karsten; Boullier, Agnes;

    2012-01-01

    Macrophage scavenger receptors appear to play a major role in the clearance of oxidized phospholipid (OxPL) products. Discrete peptide-phospholipid conjugates with the phosphatidylcholine headgroup have been shown to exhibit binding affinity for these receptors. We report the preparation of a water......-soluble, stable peptide-phospholipid conjugate (9) that possesses the necessary physical properties to enable more detailed study of the role(s) of OxPL in metabolic disease....

  12. [11C]WAY-100635 PET imaging of 5-HT1A receptor binding in patients with temporal lobe epilepsy

    To understand the role of 5-HT in human temporal lobe epilepsy, here we measured 5-HT1A receptor binding potential by positron emission tomography (PET) with [carbonyl-11C]WAY100635, a selective 5-HT1A receptor antagonist, in patients with temporal lobe epilepsy and normal controls. Twelve patients with temporal lobe epilepsy and seventeen healthy controls participated in the study. For each subject, we conducted PET and magnetic resonance imaging (MRI), by which we measured the 5-HT1A receptor binding potential, the R1-value, a relative indicator of cerebral blood flow in regions of interest, and the volume of gray matter. Patients with temporal lobe epilepsy showed significantly reduced 5-HT1A receptor binding potential in the temporal lobe. The laterality of the reduction was coincided with the epileptogenic foci estimated by a scalp electroencephalography (EEG). In contrast, the R1-value and gray matter volume showed no difference between the patient and control groups. Our study revealed that 5-HT1A receptor binding was reduced significantly at the epileptogenic foci. We suggest that PET imaging with [carbonyl-11C]WAY100635 is potentially a useful non-invasive method for determining the epileptogenic foci. (author)

  13. Characterization of histamine receptors in isolated pig basilar artery by functional and radioligand binding studies

    Histamine receptors in pig basilar arteries were investigated in vitro by radioligand binding assays and by measuring the contractile and relaxant responses to histamine. Histamine and 2-pyridyethylamine (H1-agonist) induced concentration-dependent contractions, whereas impromidine (H2-agonist) induced concentration-dependent relaxations. These responses were independent of the presence of endothelial cells. Diphenhydramine (H1-antagonist) partially reversed the histamine-induced contractions to relaxations. Cimetidine (Hα2-antagonist) potentiated the contraction in a concentration-dependent manner. In the presence of cimetidine, the pEC50 value of histamine for the contraction was 6.30, and diphenhydramine competitively antagonized the histamine-induced contractions (pA2, 7.77). In the presence of diphenhydramine, the pEC50 value of histamine for the relaxation was 5.93, and cimetidine competitively antagonized the histamine-induced relaxations (pA2, 6.62). In the binding studies, the Kd value of [3H]mepyramine was 2.1 nM and the Bmax value was 95.6 fmol/mg protein. A competition experiment with diphenhydramine showed that the pKi value (7.51) was similar to the pA2 value. The Kd value for [3H]cimetidine was 126.0 nM and the Bmax value was 459.8 fmol/mg protein. The pKd (6.90) for [3H]cimetidine was similar to the pA2 for cimetidine. The Hill coefficients for these experiments were not significantly different from unity. The present findings indicate that the number of H1-receptors, in terms of the Bmax value for [3H]mepyramine, is smaller than that of H2-receptors, in terms of the Bmax value for [3H]cimetidine. However, the contractile response to histamine is predominantly mediated through stimulation of H1-receptors on vascular smooth muscle cells in pig basilar artery

  14. Distinctive receptor binding properties of the surface glycoprotein of a natural Feline Leukemia Virus isolate with unusual disease spectrum

    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.

  15. Kinetic and structural analysis of mutant CD4 receptors that are defective in HIV gp120 binding

    Wu, Hao; Myszka, David G.; Tendian, Susan W.; Brouillette, Christie G.; Sweet, Ray W.; Chaiken, Irwin M.; Hendrickson, Wayne A.

    1996-01-01

    The T-cell antigen coreceptor CD4 also serves as the receptor for the envelope glycoprotein gp120 of HIV. Extensive mutational analysis of CD4 has implicated residues from a portion of the extracellular amino-terminal domain (D1) in gp120 binding. However, none of these proteins has been fully characterized biophysically, and thus the precise effects on molecular structure and binding interactions are unknown. In the present study, we produced soluble versions of three...

  16. Biosensor-Based Approach Identifies Four Distinct Calmodulin-Binding Domains in the G Protein-Coupled Estrogen Receptor 1

    Tran, Quang-Kim; VerMeer, Mark

    2014-01-01

    The G protein-coupled estrogen receptor 1 (GPER) has been demonstrated to participate in many cellular functions, but its regulatory inputs are not clearly understood. Here we describe a new approach that identifies GPER as a calmodulin-binding protein, locates interaction sites, and characterizes their binding properties. GPER coimmunoprecipitates with calmodulin in primary vascular smooth muscle cells under resting conditions, which is enhanced upon acute treatment with either specific liga...

  17. The role of Arg(78) in the metabotropic glutamate receptor mGlu(1) for agonist binding and selectivity

    Jensen, Anders A.; Sheppard, P O; O'Hara, P J; Krogsgaard-Larsen, P; Bräuner-Osborne, H

    The metabotropic glutamate receptors belong to family C of the G-protein coupled receptor superfamily. These receptors all possess large extracellular amino terminal domains, where agonist binding takes place. We have previously constructed a molecular model of the amino terminal domain of the m...... leucine or glutamate completely knocks out [3H]quisqualic acid binding to the receptor. The constructed mutants, R78L and R78E, have also been characterized in a inositol phosphate assay. Here, the potency of (S)-glutamic acid and (S)-quisqualic acid was reduced 1000- and 100-fold, respectively, on R78L...... compared to the wild type (WT) receptor. (S)-Quisqualic acid was as potent on mutant R78E as it was on R78L, whereas (S)-glutamic acid was unable to activate R78E significantly at concentrations up to 10 mM. In conclusion, Arg(78) appears to be essential for agonist binding to the mGlu(1) receptor, most...

  18. Genetic induction of the gastrin releasing peptide receptor on tumor cells for radiolabeled peptide binding

    Purpose/Objective: To improve upon existing radioimmunotherapy (RAIT) approaches, we have devised a strategy to genetically induce high levels of new membrane-associated receptors on human cancer cells targetable by radiolabeled peptides. In this context, we report successful adenoviral-mediated transduction of tumor cells to express the murine gastrin releasing peptide receptor (mGRPr) as demonstrated by125 I-labeled bombesin binding. Materials and Methods: To demonstrate the feasibility of our strategy and to provide rapid proof of principle, we constructed a plasmid encoding the mGRPr gene. We cloned the mGRPr gene into the adenoviral shuttle vector pACMVpLpARS+ (F. Graham). We then utilized the methodology of adenovirus-polylysine-mediated transfection (AdpLmGRPr) to accomplish transient gene expression of mGRPr in two human cancer cell lines including A427 non-small cell lung cancer cells and HeLa cervical cancer cells. Murine GRPr expression was then measured by a live-cell binding assay using 125I-labeled bombesin. In order to develop this strategy further, it was necessary to construct a vector that would be more efficient for in vivo transduction. In this regard, we constructed a recombinant adenoviral vector (AdCMVGRPr) encoding the mGRPr under the control of the CMV promoter based on in vivo homologous recombination methods. The recombinant shuttle vector containing mGRPr was co-transfected with the adenoviral rescue plasmid pJM17 into the E1A trans complementing cell line 293 allowing for derivation of replication-incompetent, recombinant adenoviral vector. Individual plaques were isolated and subjected to two further rounds of plaque purification. The identity of the virus was confirmed at each step by PCR employing primers for mGRPr. The absence of wild-type adenovirus was confirmed by PCR using primers to the adenoviral E1A gene. SKOV3.ip1 human ovarian cancer cells and MDA-MB-231 human breast cancer cells were transduced in vitro with AdCMVGRPr at

  19. In vivo binding of [{sup 11}C]nemonapride to sigma receptors in the cortex and cerebellum

    Ishiwata, Kiichi E-mail: ishiwata@pet.tmig.or.jp; Senda, Michio

    1999-08-01

    Radiolabeled nemonapride (NEM, YM-09151-2) is widely used as a representative dopamine D{sub 2}-like receptor ligand in pharmacological and neurological studies, and {sup 11}C-labeled analog ([{sup 11}C]NEM) has been developed for positron emission tomography (PET) studies. The aim of this study was to evaluate whether [{sup 11}C]NEM binds in vivo to sigma receptors. [{sup 11}C]NEM and one of six dopamine D{sub 2}-like receptor ligands or seven sigma receptor ligands were co-injected into mice, and the regional brain uptake of [{sup 11}C]NEM was measured by a tissue dissection method. The striatal uptake of [{sup 11}C]NEM was reduced by D{sub 2}-like receptor ligands, NEM, haloperidol, (+)-butaclamol, raclopride, and sulpiride, but not by a D{sub 4} receptor ligand clozapine. In the cortex and cerebellum the uptake was also reduced by D{sub 2}-like receptor ligands with affinity for sigma receptors, but not by raclopride. Although none of seven sigma receptor ligands, SA6298, N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine hydrochloride (NE-100), (+)-pentazocine, R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane hydrochloride ([-]-PPAP), (-)-pentazocine, R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ([+]-3-PPP), and (+)-N-allylnormetazocine hydrochloride ([+]-SKF 10047), blocked the striatal uptake, five of them with relatively higher affinity significantly reduced the [{sup 11}C]NEM uptake by the cortex, and four of them reduced that by the cerebellum. We concluded that [{sup 11}C]NEM binds in vivo not only to dopamine D{sub 2}-like receptors in the striatum but also to sigma receptors in other regions such as cortex and cerebellum.

  20. Crystallization and preliminary X-ray analysis of the human androgen receptor ligand-binding domain with a coactivator-like peptide and selective androgen receptor modulators

    The human androgen receptor ligand-binding domain has been crystallized as a ternary complex with a coactivator-like undecapeptide and two different synthetic ligands. The ligand-binding domain of the human androgen receptor has been cloned, overproduced and crystallized in the presence of a coactivator-like 11-mer peptide and two different nonsteroidal ligands. The crystals of the two ternary complexes were isomorphous and belonged to space group P212121, with one molecule in the asymmetric unit. They diffracted to 1.7 and 1.95 Å resolution, respectively. Structure determination of these two complexes will help in understanding the mode of binding of selective nonsteroidal androgens versus endogenous steroidal ligands and possibly the origin of their tissue selectivity