Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

A family of Ran binding proteins that includes nucleoporins.

Ran, a small nuclear GTP binding protein, is essential for the translocation of nuclear proteins through the nuclear pore complex. We show that several proteins, including the Saccharomyces cerevisiae Nup2p and Caenorhabditis elegans F59A2.1 nucleoporins, contain domains similar to the previously characterized murine Ran binding protein (RBP, termed RBP1). To test the significance of this similarity, we have used the corresponding domains of Nup2p and a putative S. cerevisiae RBP in Ran binding assays and the yeast two-hybrid system. Both proteins bind S. cerevisiae Ran, but only the putative S. cerevisiae RBP binds human Ran. Two-hybrid analysis revealed Ran-Ran interactions and that yeast and human Rans can interact. These data identify Nup2p as a target for Ran in the nuclear pore complex, suggesting a direct role for it in nuclear-cytoplasmic transport. We discuss the possibility that proteins harboring Ran binding domains link the Ran GTPase cycle to specific functions in the nucleus.

Chimeric plant calcium/calmodulin-dependent protein kinase gene with a neural visinin-like calcium-binding domain.

Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca2+ and Ca2+/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca(2+)-binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca(2+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca2+/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca(2+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approximately 56 kDa) binds calmodulin in a Ca(2+)-dependent manner. Furthermore, 45Ca-binding assays revealed that CCaMK directly binds Ca2+. The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca2+ signaling in plants.

Ratiometric and nonratiometric Ca2+ indicators for the assessment of intracellular free Ca2+ in a breast cancer cell line using a fluorescence microplate reader

Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading. (C) 2003 Elsevier B.V. All rights reserved.

Sorting nexin 5 is localized to a subdomain of the early endosomes and is recruited to the plasma imembrane following EGF stimulation

Sorting nexins are a large family of proteins that contain the phosphoinositide-binding Phox homology (PX) domain. A number of sorting nexins are known to bind to PtdIns(3)P, which mediates their localization to membranes of the endocytic pathway. We show here that sorting nexin 5 (SNX5) can be recruited to two distinct membrane compartments. In non-stimulated cells, the PX domain was independently targeted to endosomal structures and colocalized with full-length SNX5. The membrane binding of the PX domain was inhibited by the PI 3-kinase inhibitor, wortmannin. Although SNX5 colocalized with a fluid-phase marker and was found predominantly within a PtdIns(3)P-rich endosomal domain, very little colocalization was observed between SNX5 and the PtdIns(3)P-binding protein, EEA1. Using liposome-based binding assays, we have shown that the PX domain of SNX5 interacts not only with PtdIns(3)P but also with PtdIns(3,4)P-2. In response to EGF stimulation, either the SNX5-PX domain or full-length SNX5 was rapidly recruited to the plasma membrane. The localization of SNX1, which does not bind PtdIns(3,4)P-2, was unaffected by EGF signalling. Therefore, SNX5 is localized to a subdomain of the early endosome distinct from EEA1 and, following EGF stimulation and elevation of PtdIns(3,4)P-2, is also transiently recruited to the plasma membrane. These results indicate that SNX5 may have functions not only associated with endosomal sorting but also with the phosphoinositide-signalling pathway.

Increased site 1 affinity improves biopotency of porcine growth hormone - Evidence against diffusion dependent receptor dimerization

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp(104) and Trp(169) in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys(165)), which in addition to His(169), restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.

Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions. (c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer ( FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

C/EBP delta and C/EBP gamma bind the CCAAT-box in the human beta-globin promoter and modulate the activity of the CACC-box binding protein, EKLF

Developmental- and tissue-specific expression of globin genes is mediated by a few key elements within the proximal promoter of each gene. DNA-binding assays previously identified NF-Y, GATA-1, C/EBP beta and C/EBP gamma as candidate regulators of beta-globin transcription via the CCAAT-box, a promoter element situated between CACC- and TATA-boxes. We have identified C/EBP delta as an additional beta-globin CCAAT-box binding protein. In reporter assays, we show that C/EBP delta can co-operate with EKLF, a CACC-box binding protein, to activate the beta-globin promoter, whereas C/EBP gamma inhibits the transcriptional activity of EKLF in this assay. (c) 2005 Elsevier B.V. All rights reserved.

Synthesis and biological properties of 2-phenylbenzothiazole derivatives

2-Phenylbenzothiazoles have structural similarities to the antioestrogenic 2-phenylindole, zindoxifene and to the oestrogenic isoflavone, genistein which also inhibits tyrosine kinases. Hydroxylated 2-phenylbenzothiazole derivatives were therefore produced and tested for oestrogenic and tyrosine kinase inhibitory activity. Synthesis of methoxy substituted 2-phenylbenzothiazoles was via the Jacobson method, demethylation being effected by boron tribromide at -70oC. Three amino substituted 2-phenylbenzothiazoles were also synthesised and tested for activity. Data is presented for oestrogen receptor binding activity, aromatase inhibitory activity, epidermal growth factor receptor tyrosine kinase (EGFRTK) inhibitory activity and cytotoxicity to ANN-1, 3T3, MCF-7 and WIDR cells. Oestrogen receptor binding affinity (RBA) was shown by five of the nine compounds tested. 2-(4-hydroxy)-6-hydroxybenzo-thiazole was the most active of the benzothiazoles tested (RBA 0.7). This is low but comparable to that of genistein. EGFRTK inhibitory activity was shown by four of the six benzothiazole derivatives tested; activity was comparable to that of genistein. Cytotoxicity assays have shown no selective toxicity of 2-phenylbenzothiazoles to any of the cell lines tested. Toxicity to MCF-7 cells was similar to that for other cell lines despite some compounds showing oestrogen receptor binding capacity. Amino-substituted 2-phenylbenzothiazoles showed selective toxicity towards transformed ANN-1 cells compared to normal 3T3 cells but the mechanism of this selectivity has not been established. Molecular modelling techniques, including CHEM-X, QUANTA and MOPAC were used to compare known ATP-competitive tyrosine kinase inhibitors with a model of ATP built from the crystal structure of the ATP-phosphoglycerate kinase complex. Structural features thought to be important to kinase inhibition were found and used to suggest further 2-phenylbenzothiazole analogues which may have improved activity.

Targeting the glycoprotein 130 receptor subunit to control pain and inflammation

The glycoprotein 130 (gp130) is a shared signal-transducing-membrane-associated receptor for several hematopoietic cytokines. Its activation is implicated in pain and in a variety of diseases via signaling of proinflammatory cytokines. These include interleukin-6 (IL-6) subfamily cytokines, many of which play important roles in the pathogenesis of diseases such as rheumatoid arthritis, Castleman's disease, and Kaposi's sarcoma. Several strategies have been developed to block gp130-receptor-mediated signaling. These include the application of monoclonal antibodies, the creation of mutant form(s) of the gp130 with increased binding affinity for such ligands as IL-6/sIL-6R complex, and the generation of antagonists by selective mutagenesis of the specific cytokine/gp130 receptor binding site(s). Other strategies include targeting gp130-mediated signaling pathways such as that involving signal transducer and activator of transcription-3. This review provides a summary of the latest research pertaining to the role of gp130 in the pathogenesis of inflammatory and other diseases in which the gp130 receptor is implicated. An overview of antagonists targeting the gp130 receptor is included with particular emphasis on their mechanism of action and their limitations and potential for therapeutic application.

Synthesis and evaluation of N-(3-oxo-2,3-dihydro-1H-pyrazol-4-yl)-1H-indole-carboxamides as cholecystokinin antagonists

The structure-activity relationship optimization of the pyrazoline template 3a resulted in novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides 4a-4e. These non-peptidal CCK ligands have been shown to act as potent CCK 1 ligands in a [125]I-CCK-8 receptor binding assay. The best amides (4c and 4d) of this series displayed an IC50 of 20/25 CCK 1 for the CCK 1 receptor. In a subsequent in-vivo evaluation using various behaviour pharmacological assays, an anxiolytic effect of these novel 3-oxo-1,2-diphenyl-2,3-dihydro-1H-pyrazol-4-yl)-indole carboxamides was found at high doses in the elevated plus-maze. In the despair swimming test, a model for testing antidepressants, an ED50 of 0.33/0.41 mg kg -1 was determined for amide 4c/4d and the antidepressant effect had a magnitude comparable to desimipramine. © 2006 The Authors.

Efeitos in vivo e in vitro de agonistas parciais do receptor NOP: implicações para o tratamento da ansiedade, depressão e mania

Introduction: This study aimed to investigate the effects of the two peptide NOP partial agonists (UFP-113 and [F/G]N/OFQ(1-13)NH2) and the non peptide NOP partial agonist (AT-090) in the mouse emotional behavior as well as in the intracellular transduction pathways following the receptor binding. Methods: Male Swiss or CD-1 mice were used in this study together with NOP(+/+) and NOP(-/-) mice. The elevated plus maze (EPM) was used to evaluate the effects of compounds on anxiety-like behaviors. Diazepam and the NOP agonists, N/OFQ and Ro 65-6570, were used as positive controls in the EPM. NOP(+/+) and NOP(-/-) mice were used to evaluate the selectivity of those compounds that induced anxiolytic-like behaviors. The forced swim test (FST) was used to evaluate the effects of compounds on depressive-like behaviors. Nortriptyline and the NOP antagonists, UFP-101 and SB-612111, were used as positive controls in the FST. The effects of N/OFQ, UFP-101, SB-612111, UFP-113, [F/G]N/OFQ(1-13)NH2, and AT-090 were assessed in the methylphenidate-induced hyperlocomotion (MIH) test; in this assay valproate was used as positive control. The G protein and β-arrestin 2 transduction pathways of NOP receptor agonists (N/OFQ and Ro 65-6570), antagonist (UFP-101), and partial agonists (UFP-113, [F/G]N/OFQ(1-13)NH2, and AT-090) were also evaluated using an innovative assay that measures a bioluminescence resonance energy transfer process. For this, cell lines permanently co-expressing the NOP receptor coupled to luciferase (energy donor), and green fluorescent protein (energy acceptor) coupled to one of the effector proteins (G protein or β-arrestin 2) were used. Results: Diazepam (1 mg/kg), N/OFQ (1 nmol), Ro 65-6570 (0.1 mg/kg), and AT-090 (0.01 mg/kg) induced anxiolytic-like effect in mice in the EPM. The effects of Ro 65-6570 and AT-090 were selective to NOP receptor. UFP-113 (0.01-1 nmol) and [F/G]N/OFQ(1-13)NH2 (0.1-3 nmol) were inactive in the EPM. In the FST, nortriptyline (30 mg/kg), UFP-101 (10 nmol), SB-612111 (10 mg/kg), UFP-113 (0.01 and 0.1 nmol), and [F/G]N/OFQ(1-13)NH2 (0.3 and 1 nmol) induced antidepressant-like effects, while AT-090 (0.001-0.1 mg/kg) was inactive in this assay. The effects of UFP-113 and [F/G]N/OFQ(1-13)NH2 were selective to NOP receptor. Valproate (400 mg/kg) counteracted methylphenidate (MPH, 10 mg/kg)-induced hyperlocomotion in mice in the open field. N/OFQ (1 nmol), UFP-113 (0.01-0.1 nmol), and [F/G]N/OFQ(1-13)NH2 (1 nmol) were also able to reduce the MPH-induced hyperlocomotion, without changing the locomotor activity per se. The effect of UFP-113 was selective to NOP receptor. The UFP-101 (10 nmol), SB-612111 (10 mg/kg), and AT-090 (0.001-0.03 mg/kg) did not change the hyperlocomotor effect of methylphenidate. In vitro, N/OFQ and Ro 65-6570 behaved as NOP full agonists for G-protein and β-arrestin 2 pathways. AT-090 behaved as NOP receptor partial agonist for both transduction pathways, while UFP-113 and [F/G]N/OFQ(1-13)NH2 behaved as partial agonists and antagonists of NOP receptor for NOP/G protein and NOP/β-arrestin 2, respectively. UFP-101 behaved as NOP receptor antagonist for both transduction pathways. Conclusion: NOP ligands producing same effects on NOP/G protein interaction (partial agonism), but with opposite effects on β-arrestin 2 recruitment (partial agonism vs antagonism), can promote different in vivo effects on anxiety and mood as it was observed in the behavioral tests. This work corroborates the potential of NOP receptor as an innovative pharmacological target for the treatment of emotional disorders.

Efeitos in vivo e in vitro de agonistas parciais do receptor NOP: implicações para o tratamento da ansiedade, depressão e mania

Introduction: This study aimed to investigate the effects of the two peptide NOP partial agonists (UFP-113 and [F/G]N/OFQ(1-13)NH2) and the non peptide NOP partial agonist (AT-090) in the mouse emotional behavior as well as in the intracellular transduction pathways following the receptor binding. Methods: Male Swiss or CD-1 mice were used in this study together with NOP(+/+) and NOP(-/-) mice. The elevated plus maze (EPM) was used to evaluate the effects of compounds on anxiety-like behaviors. Diazepam and the NOP agonists, N/OFQ and Ro 65-6570, were used as positive controls in the EPM. NOP(+/+) and NOP(-/-) mice were used to evaluate the selectivity of those compounds that induced anxiolytic-like behaviors. The forced swim test (FST) was used to evaluate the effects of compounds on depressive-like behaviors. Nortriptyline and the NOP antagonists, UFP-101 and SB-612111, were used as positive controls in the FST. The effects of N/OFQ, UFP-101, SB-612111, UFP-113, [F/G]N/OFQ(1-13)NH2, and AT-090 were assessed in the methylphenidate-induced hyperlocomotion (MIH) test; in this assay valproate was used as positive control. The G protein and β-arrestin 2 transduction pathways of NOP receptor agonists (N/OFQ and Ro 65-6570), antagonist (UFP-101), and partial agonists (UFP-113, [F/G]N/OFQ(1-13)NH2, and AT-090) were also evaluated using an innovative assay that measures a bioluminescence resonance energy transfer process. For this, cell lines permanently co-expressing the NOP receptor coupled to luciferase (energy donor), and green fluorescent protein (energy acceptor) coupled to one of the effector proteins (G protein or β-arrestin 2) were used. Results: Diazepam (1 mg/kg), N/OFQ (1 nmol), Ro 65-6570 (0.1 mg/kg), and AT-090 (0.01 mg/kg) induced anxiolytic-like effect in mice in the EPM. The effects of Ro 65-6570 and AT-090 were selective to NOP receptor. UFP-113 (0.01-1 nmol) and [F/G]N/OFQ(1-13)NH2 (0.1-3 nmol) were inactive in the EPM. In the FST, nortriptyline (30 mg/kg), UFP-101 (10 nmol), SB-612111 (10 mg/kg), UFP-113 (0.01 and 0.1 nmol), and [F/G]N/OFQ(1-13)NH2 (0.3 and 1 nmol) induced antidepressant-like effects, while AT-090 (0.001-0.1 mg/kg) was inactive in this assay. The effects of UFP-113 and [F/G]N/OFQ(1-13)NH2 were selective to NOP receptor. Valproate (400 mg/kg) counteracted methylphenidate (MPH, 10 mg/kg)-induced hyperlocomotion in mice in the open field. N/OFQ (1 nmol), UFP-113 (0.01-0.1 nmol), and [F/G]N/OFQ(1-13)NH2 (1 nmol) were also able to reduce the MPH-induced hyperlocomotion, without changing the locomotor activity per se. The effect of UFP-113 was selective to NOP receptor. The UFP-101 (10 nmol), SB-612111 (10 mg/kg), and AT-090 (0.001-0.03 mg/kg) did not change the hyperlocomotor effect of methylphenidate. In vitro, N/OFQ and Ro 65-6570 behaved as NOP full agonists for G-protein and β-arrestin 2 pathways. AT-090 behaved as NOP receptor partial agonist for both transduction pathways, while UFP-113 and [F/G]N/OFQ(1-13)NH2 behaved as partial agonists and antagonists of NOP receptor for NOP/G protein and NOP/β-arrestin 2, respectively. UFP-101 behaved as NOP receptor antagonist for both transduction pathways. Conclusion: NOP ligands producing same effects on NOP/G protein interaction (partial agonism), but with opposite effects on β-arrestin 2 recruitment (partial agonism vs antagonism), can promote different in vivo effects on anxiety and mood as it was observed in the behavioral tests. This work corroborates the potential of NOP receptor as an innovative pharmacological target for the treatment of emotional disorders.

Counteractive effects of antenatal glucocorticoid treatment on D1 receptor modulation of spatial working memory

RATIONALE: Antenatal exposure to the glucocorticoid dexamethasone dramatically increases the number of mesencephalic dopaminergic neurons in rat offspring. However, the consequences of this expansion in midbrain dopamine (DA) neurons for behavioural processes in adulthood are poorly understood, including working memory that depends on DA transmission in the prefrontal cortex (PFC). OBJECTIVES: We therefore investigated the influence of antenatal glucocorticoid treatment (AGT) on the modulation of spatial working memory by a D1 receptor agonist and on D1 receptor binding and DA content in the PFC and striatum. METHODS: Pregnant rats received AGT on gestational days 16-19 by adding dexamethasone to their drinking water. Male offspring reared to adulthood were trained on a delayed alternation spatial working memory task and administered the partial D1 agonist SKF38393 (0.3-3 mg/kg) by systemic injection. In separate groups of control and AGT animals, D1 receptor binding and DA content were measured post-mortem in the PFC and striatum. RESULTS: SKF38393 impaired spatial working memory performance in control rats but had no effect in AGT rats. D1 binding was significantly reduced in the anterior cingulate cortex, prelimbic cortex, dorsal striatum and ventral pallidum of AGT rats compared with control animals. However, AGT had no significant effect on brain monoamine levels. CONCLUSIONS: These findings demonstrate that D1 receptors in corticostriatal circuitry down-regulate in response to AGT. This compensatory effect in D1 receptors may result from increased DA-ergic tone in AGT rats and underlie the resilience of these animals to the disruptive effects of D1 receptor activation on spatial working memory.