23 results on '"Mark H. Pausch"'
Search Results
2. Identification of MK-8133: An orexin-2 selective receptor antagonist with favorable development properties
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Christina N. DiMarco, Anthony L. Gotter, Alan T. Savitz, Joanne Stevens, Joseph G. Bruno, Tamara D. Cabalu, John J. Renger, Jason W. Skudlarek, Pamela L. Tannenbaum, Paul J. Coleman, Scott D. Kuduk, Joseph Brunner, Susan L. Garson, Christopher J. Winrow, Julie A. O'Brien, Charles M. Harrell, and Mark H. Pausch
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Filorexant ,medicine.drug_class ,Clinical Biochemistry ,Pharmaceutical Science ,Pharmacology ,Biochemistry ,Mice ,Structure-Activity Relationship ,chemistry.chemical_compound ,Dogs ,Piperidines ,Orexin Receptors ,Sleep Initiation and Maintenance Disorders ,mental disorders ,Drug Discovery ,medicine ,Animals ,Benzamide ,Molecular Biology ,G protein-coupled receptor ,Organic Chemistry ,Antagonist ,Triazoles ,Receptor antagonist ,Orexin receptor ,Rats ,Orexin ,Pyrimidines ,chemistry ,Molecular Medicine ,Orexin Receptor Antagonists ,Antagonism ,Half-Life ,Protein Binding ,medicine.drug - Abstract
Antagonism of orexin receptors has shown clinical efficacy as a novel paradigm for the treatment of insomnia and related disorders. Herein, molecules related to the dual orexin receptor antagonist filorexant were transformed into compounds that were selective for the OX2R subtype. Judicious selection of the substituents on the pyridine ring and benzamide groups led to 6b; which was highly potent, OX2R selective, and exhibited excellent development properties.
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- 2015
3. Development of an Improved IP1 Assay for the Characterization of 5-HT2C Receptor Ligands
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Mark H. Pausch, John Dunlop, Stanley Nawoschik, Jean Y. Zhang, Dianne Kowal, and Ravikumar Peri
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biology ,Phospholipase C ,Chemistry ,Inositol Phosphates ,Chinese hamster ovary cell ,Stimulation ,CHO Cells ,Ligands ,biology.organism_classification ,Molecular biology ,Cricetulus ,Förster resonance energy transfer ,Biochemistry ,Cricetinae ,Drug Discovery ,Fluorescence Resonance Energy Transfer ,Receptor, Serotonin, 5-HT2C ,Animals ,Humans ,Molecular Medicine ,Calcium ,Serotonin ,Receptor ,5-HT receptor - Abstract
The 5-hydroxytryptamine 2C (5-HT(2C)) receptor is a member of the serotonin 5-HT(2) subfamily of G-protein-coupled receptors signaling predominantly via the phospholipase C (PLC) pathway. Stimulation of phosphoinositide (PI) hydrolysis upon 5-HT(2C) receptor activation is traditionally assessed by measuring inositol monophosphate (IP(1)) using time-consuming and labor-intensive anion exchange radioactive assays. In this study, we have developed and optimized a cellular IP(1) assay using homogeneous time-resolved fluorescence (HTRF), a fluorescence resonance energy transfer (FRET)-based technology (Cisbio; Gif sur Yvette, France). The measurement is simple to carry out without the cumbersome steps associated with radioactive assays and may therefore be used as an alternative tool to evaluate PI hydrolysis activated by 5-HT(2C) agonists. In Chinese hamster ovary (CHO) cells stably expressing 5-HT(2C) receptors, characterization of 5-HT(2C) agonists with the HTRF platform revealed a rank order of potency (EC(50), nM) comparable to that from intracellular calcium mobilization studies measured by the fluorometric imaging plate reader (FLIPR). A similar rank order of potency was seen with conventional radioactive PI assay with the exception of 5-HT. Lastly, the new assay data correlated better with agonist-induced calcium responses in FLIPR (R(2) = 0.78) than with values determined by radioactive IP(1) method (R(2) = 0.64). Our study shows that the HTRF FRET-based assay detects IP(1) with good sensitivity and may be streamlined for high-throughput (HTS) applications.
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- 2010
4. ADX47273 [S-(4-Fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]-oxadiazol-5-yl]-piperidin-1-yl}-methanone]: A Novel Metabotropic Glutamate Receptor 5-Selective Positive Allosteric Modulator with Preclinical Antipsychotic-Like and Procognitive Activities
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Steven M. Grauer, Peter J. Atkinson, Michael A. Olsen, Cody Kelley, Deborah L. Smith, Margaret Lai, Guoming Zhang, Feng Liu, Sharon Rosenzweig-Lipson, Chad E. Beyer, Michael Popiolek, Adam M. Gilbert, Mark Day, Karen L. Marquis, Radka Graf, Rachel Navarra, Claudine Pulicicchio, Farhana Pruthi, Xavier Z. Khawaja, Evguenia Kouranova, Sheree F. Logue, Tom A. Comery, Caitlin Wantuch, Mark H. Pausch, and Nicholas J. Brandon
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Allosteric modulator ,Receptor, Metabotropic Glutamate 5 ,Allosteric regulation ,Drug Evaluation, Preclinical ,Prefrontal Cortex ,CDPPB ,Pharmacology ,Receptors, Metabotropic Glutamate ,Hippocampus ,Cell Line ,Cognition ,Allosteric Regulation ,Piperidines ,Dopamine ,Avoidance Learning ,medicine ,Animals ,Humans ,Phencyclidine ,Brain Chemistry ,Oxadiazoles ,Dose-Response Relationship, Drug ,Metabotropic glutamate receptor 5 ,Chemistry ,Glutamate receptor ,Rats ,Metabotropic glutamate receptor ,Molecular Medicine ,Antipsychotic Agents ,medicine.drug - Abstract
Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca(2+) assays (EC(50) = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 microM) and competed for binding of [(3)H]2-methyl-6-(phenylethynyl)pyridine (K(i) = 4.3 microM), but not [(3)H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.
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- 2008
5. Characterization of Gpr101 expression and G-protein coupling selectivity
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Eugene Tseng, Ying Sun, Maria Blatcher, Jeremy Johnson, Lynn Zhang, Noel Taylor, Sreekumar Raman Kodangattil, Janet E. Paulsen, Stan P. Nawoschik, Brian Bates, David C. Kopsco, Angela Kramer, Hui Min Chen, Qin Shan, and Mark H. Pausch
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Gs alpha subunit ,G protein ,Molecular Sequence Data ,Gene Expression ,Nerve Tissue Proteins ,Biology ,Transfection ,Models, Biological ,Cell Line ,Receptors, G-Protein-Coupled ,Mice ,GTP-Binding Proteins ,Genes, Reporter ,Two-Hybrid System Techniques ,Gene expression ,Cyclic AMP ,Animals ,Humans ,Genetic Testing ,Cloning, Molecular ,Molecular Biology ,Gene ,In Situ Hybridization ,Gene Library ,G protein-coupled receptor ,Reporter gene ,Messenger RNA ,General Neuroscience ,HEK 293 cells ,Brain ,Chromosome Mapping ,Blotting, Northern ,Molecular biology ,Neurology (clinical) ,Developmental Biology - Abstract
This report describes the identification and characterization of the murine orphan GPCR, Gpr101. Both human and murine genes were localized to chromosome X. Similar to its human ortholog, murine Gpr101 mRNA was detected predominantly in the brain within discrete nuclei. A knowledge-restricted hidden Markov model-based algorithm, capable of accurately predicting G-protein coupling selectivity, indicated that both human and murine GPR101 were likely coupled to Gs. This prediction was supported by the elevation of cyclic AMP levels and the activation of a cyclic AMP response element-luciferase reporter gene in HEK293 cells over-expressing human GPR101. Consistent with this, over-expression of human GPR101 in a yeast-based system yielded an elevated, agonist-independent reporter gene response in the presence of a yeast chimeric Gαs protein. These results indicate that GPR101 participates in a potentially wide range of activities in the CNS via modulation of cAMP levels.
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- 2006
6. Development of a Yeast Bioassay to Characterize G Protein-coupled Receptor Kinases
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Mark H. Pausch, Lorena A. Kallal, Jeffrey L. Benovic, and Beth Noble
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G protein-coupled receptor kinase ,Growth factor receptor ,G-Protein-Coupled Receptor Kinase 5 ,Biochemistry ,Autophosphorylation ,5-HT5A receptor ,Cell Biology ,Biology ,Interleukin-13 receptor ,Molecular Biology ,Protease-activated receptor 2 ,Insulin-like growth factor 1 receptor - Abstract
G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.
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- 2003
7. Functional expression of M1, M3 and M5 muscarinic acetylcholine receptors in yeast
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Evi Kostenis, Jürgen Wess, Clarice Schmidt, Isolde Erlenbach, Mark H. Pausch, and Fadi F. Hamdan
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Cellular and Molecular Neuroscience ,Biochemistry ,Muscarinic acetylcholine receptor M5 ,Muscarinic acetylcholine receptor M3 ,Muscarinic acetylcholine receptor M2 ,Class C GPCR ,Muscarinic acetylcholine receptor M1 ,Biology ,Receptor ,Molecular biology ,Rhodopsin-like receptors ,G protein-coupled receptor - Abstract
The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.
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- 2001
8. Single Amino Acid Substitutions and Deletions That Alter the G Protein Coupling Properties of the V2 Vasopressin Receptor Identified in Yeast by Receptor Random Mutagenesis
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Danielle Raufaste, Clarice Schmidt, Mark H. Pausch, Evi Kostenis, Claudine Serradeil-Le Gal, Jürgen Wess, Mark E. Dumont, and Isolde Erlenbach
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Models, Molecular ,Receptors, Vasopressin ,Saccharomyces cerevisiae Proteins ,Molecular Sequence Data ,Saccharomyces cerevisiae ,Biology ,Polymerase Chain Reaction ,Biochemistry ,Gamma-aminobutyric acid receptor subunit alpha-1 ,Protein Structure, Secondary ,Animals ,Humans ,5-HT5A receptor ,Amino Acid Sequence ,Cloning, Molecular ,GABBR1 ,Molecular Biology ,Nuclear receptor co-repressor 1 ,Protease-activated receptor 2 ,DNA Primers ,Gene Library ,Sequence Deletion ,G protein-coupled receptor ,G protein-coupled receptor kinase ,Base Sequence ,Sequence Homology, Amino Acid ,Liver receptor homolog-1 ,Cell Membrane ,Cell Biology ,Heterotrimeric GTP-Binding Proteins ,GTP-Binding Protein alpha Subunits ,Arginine Vasopressin ,Kinetics ,Protein Subunits ,Amino Acid Substitution ,Oligodeoxyribonucleotides ,Mutagenesis, Site-Directed ,GTP-Binding Protein alpha Subunits, Gq-G11 ,Cattle ,Sequence Alignment - Abstract
To facilitate structure-function relationship studies of the V2 vasopressin receptor, a prototypical G(s)-coupled receptor, we generated V2 receptor-expressing yeast strains (Saccharomyces cerevisiae) that required arginine vasopressin-dependent receptor/G protein coupling for cell growth. V2 receptors heterologously expressed in yeast were unable to productively interact with the endogenous yeast G protein alpha subunit, Gpa1p, or a mutant Gpa1p subunit containing the C-terminal G alpha(q) sequence (Gq5). In contrast, the V2 receptor efficiently coupled to a Gpa1p/G alpha(s) hybrid subunit containing the C-terminal G alpha(s) sequence (Gs5), indicating that the V2 receptor retained proper G protein coupling selectivity in yeast. To gain insight into the molecular basis underlying the selectivity of V2 receptor/G protein interactions, we used receptor saturation random mutagenesis to generate a yeast library expressing mutant V2 receptors containing mutations within the second intracellular loop. A subsequent yeast genetic screen of about 30,000 mutant receptors yielded four mutant receptors that, in contrast to the wild-type receptor, showed substantial coupling to Gq5. Functional analysis of these mutant receptors, followed by more detailed site-directed mutagenesis studies, indicated that single amino acid substitutions at position Met(145) in the central portion of the second intracellular loop of the V2 receptor had pronounced effects on receptor/G protein coupling selectivity. We also observed that deletion of single amino acids N-terminal of Met(145) led to misfolded receptor proteins, whereas single amino acid deletions C-terminal of Met(145) had no effect on V2 receptor function. These findings highlight the usefulness of combining receptor random mutagenesis and yeast expression technology to study mechanisms governing receptor/G protein coupling selectivity and receptor folding.
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- 2001
9. Functional Coupling of a Mammalian Somatostatin Receptor to the Yeast Pheromone Response Pathway
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Eileen M. Kajkowski, Bradley A. Ozenberger, J R Hadcock, Mark H. Pausch, and L A Price
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Receptors, Peptide ,G protein ,Molecular Sequence Data ,Saccharomyces cerevisiae ,Pheromones ,GTP-Binding Proteins ,Animals ,Somatostatin receptor 2 ,Somatostatin receptor 1 ,Receptors, Somatostatin ,Molecular Biology ,DNA Primers ,Delta cell ,Base Sequence ,biology ,Somatostatin receptor ,Cell Membrane ,Cell Biology ,biology.organism_classification ,Recombinant Proteins ,Yeast ,Rats ,Biochemistry ,Receptors, Mating Factor ,Biological Assay ,Signal transduction ,Somatostatin ,Cell Division ,Research Article ,Signal Transduction ,Transcription Factors - Abstract
A detailed analysis of structural and functional aspects of G-protein-coupled receptors, as well as discovery of novel pharmacophores that exert their effects on members of this class of receptors, will be facilitated by development of a yeast-based bioassay. To that end, yeast strains that functionally express the rat somatostatin receptor subtype 2 (SSTR2) were constructed. High-affinity binding sites for somatostatin ([125I-Tyr-11]S-14) comparable to those in native tissues were detected in yeast membrane extracts at levels equivalent to the alpha-mating pheromone receptor (Ste2p). Somatostatin-dependent growth of strains modified by deletion of genes encoding components of the pheromone response pathway was detected through induction of a pheromone-responsive HIS3 reporter gene, enabling cells to grow on medium lacking histidine. Dose-dependent growth responses to S-14 and related SSTR2 subtype-selective agonists that were proportional to the affinity of the ligands for SSTR2 were observed. The growth response required SSTR2, G alpha proteins, and an intact signal transduction pathway. The sensitivity of the bioassay was affected by intracellular levels of the G alpha protein. A mutation in the SST2 gene, which confers supersensitivity to pheromone, was found to significantly enhance the growth response to S-14. In sst2 delta cells, SSTR2 functionally interacted with both a chimeric yeast/mammalian G alpha protein and the yeast G alpha protein, Gpa1p; to promote growth. These yeast strains should serve as a useful in vivo reconstitution system for examination of molecular interactions of the G-protein-coupled receptors and G proteins.
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- 1995
10. D-amino acid oxidase activity is inhibited by an interaction with bassoon protein at the presynaptic active zone
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Pranab K. Chanda, Eckart D. Gundelfinger, John F. Ross, Stephen J. Moss, Mark H. Pausch, Michael Popiolek, Erik I. Charych, and Nicholas J. Brandon
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D-Amino-Acid Oxidase ,Male ,Immunoprecipitation ,Population ,D-amino acid oxidase ,Presynaptic Terminals ,Synaptic Membranes ,Nerve Tissue Proteins ,Biology ,Biochemistry ,Receptors, N-Methyl-D-Aspartate ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Glutamatergic ,Neurobiology ,Cerebellum ,Serine ,Animals ,Humans ,Receptor ,education ,Neurotransmitter ,Molecular Biology ,education.field_of_study ,Neuropeptides ,Cell Biology ,Rats ,Cytoskeletal Proteins ,Membrane protein ,chemistry ,Schizophrenia ,Presynaptic active zone - Abstract
Schizophrenia is a highly heritable neuropsychiatric disorder affecting ∼1% of the world's population. Linkage and association studies have identified multiple candidate schizophrenia susceptibility genes whose functions converge on the glutamatergic neurotransmitter system. One such susceptibility gene encoding d-amino acid oxidase (DAO), an enzyme that metabolizes the NMDA receptor (NMDAR) co-agonist d-serine, has the potential to modulate NMDAR function in the context of schizophrenia. To further investigate its cellular regulation, we sought to identify DAO-interacting proteins that participate in its functional regulation in rat cerebellum, where DAO expression is especially high. Immunoprecipitation with DAO-specific antibodies and subsequent mass spectrometric analysis of co-precipitated proteins yielded 24 putative DAO-interacting proteins. The most robust interactions occurred with known components of the presynaptic active zone, such as bassoon (BSN) and piccolo (PCLO). The interaction of DAO with BSN was confirmed through co-immunoprecipitation assays using DAO- and BSN-specific antibodies. Moreover, DAO and BSN colocalized with one another in cultured cerebellar granule cells and in synaptic junction membrane protein fractions derived from rat cerebellum. The functional consequences of this interaction were studied through enzyme assay experiments, where DAO enzymatic activity was significantly inhibited as a result of its interaction with BSN. Taking these results together, we hypothesize that synaptic d-serine concentrations may be under tight regulation by a BSN-DAO complex. We therefore predict that this mechanism plays a role in the modulation of glutamatergic signaling through NMDARs. It also furthers our understanding of the biology underlying this potential therapeutic entry point for schizophrenia and other psychiatric disorders.
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- 2011
11. Potent dihydroquinolinone dopamine D2 partial agonist/serotonin reuptake inhibitors for the treatment of schizophrenia
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Jean Zhang, Ping Zhou, Tikva Carrick, Albert J. Robichaud, Rolf Feenstra, Jan-Hendrik Reinders, Dianne Kowal, Chris G. Kruse, Martina A.W. van der Neut, Yinfa Yan, David P. Rotella, Mark H. Pausch, Margaret Lai, and Karen L. Marquis
- Subjects
Serotonin reuptake inhibitor ,Clinical Biochemistry ,Pharmaceutical Science ,Pharmacology ,Quinolones ,Biochemistry ,Partial agonist ,Reuptake ,Structure-Activity Relationship ,Dopamine receptor D2 ,Drug Discovery ,Moiety ,Animals ,Molecular Biology ,biology ,Chemistry ,Receptors, Dopamine D2 ,Organic Chemistry ,Disease Models, Animal ,Norepinephrine transporter ,Dopamine Agonists ,Receptor, Serotonin, 5-HT1A ,biology.protein ,Schizophrenia ,Molecular Medicine ,Pharmacophore ,Endogenous agonist ,Selective Serotonin Reuptake Inhibitors ,Antipsychotic Agents - Abstract
A dihydroquinolinone moiety was found to be a potent serotonin reuptake inhibitor pharmacophore when combined with certain amines. This fragment was coupled with selected D2 ligands to prepare a series of dual acting compounds with attractive in vitro profiles as dopamine D2 partial agonists and serotonin reuptake inhibitors. Structure–activity studies revealed that the linker plays a key role in contributing to D2 affinity, function, and SRI activity.
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- 2010
12. WS-50030 [7-{4-[3-(1H-inden-3-yl)propyl]piperazin-1-yl}-1,3-benzoxazol-2(3H)-one]: a novel dopamine D2 receptor partial agonist/serotonin reuptake inhibitor with preclinical antipsychotic-like and antidepressant-like activity
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Sharon Rosenzweig-Lipson, Deborah L. Smith, Feenstra Roelof W, Steven M. Grauer, Pierre Broqua, Rachel Navarra, David P. Rotella, Mark H. Pausch, Karen L. Marquis, Radka Graf, Albert J. Robichaud, Martina Van De Neut, Qian Lin, Claudine Pulicicchio, Farhana Pruthi, Wouter Goutier, Chad E. Beyer, Caitlin Wantuch, Julie A. Brennan, Zoë A. Hughes, Andrew C. McCreary, Margaret Lai, and Chris G. Kruse
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Male ,Serotonin ,medicine.medical_treatment ,Serotonin reuptake inhibitor ,Dopamine ,Microdialysis ,Drug Evaluation, Preclinical ,Mice, Inbred Strains ,CHO Cells ,Pharmacology ,Motor Activity ,Serotonin 5-HT1 Receptor Antagonists ,Transfection ,Partial agonist ,Rats, Sprague-Dawley ,Mice ,Cricetulus ,Dopamine receptor D2 ,Cricetinae ,medicine ,Serotonin 5-HT2 Receptor Antagonists ,Avoidance Learning ,Animals ,Humans ,Rats, Wistar ,Antipsychotic ,Serotonin transporter ,Benzoxazoles ,biology ,Behavior, Animal ,Chemistry ,Receptors, Dopamine D2 ,Brain ,Antidepressive Agents ,Rats ,Indenes ,Dopamine Agonists ,biology.protein ,Molecular Medicine ,Aripiprazole ,Selective Serotonin Reuptake Inhibitors ,medicine.drug ,Antipsychotic Agents ,Protein Binding - Abstract
The preclinical characterization of WS-50030 [7-{4-[3-(1H-inden-3-yl)propyl]piperazin-1-yl}-1,3-benzoxazol-2(3H)-one] is described. In vitro binding and functional studies revealed highest affinity to the D(2) receptor (D(2L) K(i), 4.0 nM) and serotonin transporter (K(i), 7.1 nM), potent D(2) partial agonist activity (EC(50), 0.38 nM; E(max), 30%), and complete block of the serotonin transporter (IC(50), 56.4 nM). Consistent with this in vitro profile, WS-50030 (10 mg/kg/day, 21 days) significantly increased extracellular 5-HT in the rat medial prefrontal cortex, short-term WS-50030 treatment blocked apomorphine-induced climbing (ID(50), 0.51 mg/kg) in a dose range that produced minimal catalepsy in mice and induced low levels of contralateral rotation in rats with unilateral substantia nigra 6-hydroxydopamine lesions (10 mg/kg i.p.), a behavioral profile similar to that of the D(2) partial agonist aripiprazole. In a rat model predictive of antipsychotic-like activity, WS-50030 and aripiprazole reduced conditioned avoidance responding by 42 and 55% at 10 mg/kg, respectively. Despite aripiprazole's reported lack of effect on serotonin transporters, long-term treatment with aripiprazole or WS-50030 reversed olfactory bulbectomy-induced hyperactivity at doses that did not reduce activity in sham-operated rats, indicating antidepressant-like activity for both compounds. Despite possessing serotonin reuptake inhibitory activity in addition to D(2) receptor partial agonism, WS-50030 displays activity in preclinical models predictive of antipsychotic- and antidepressant efficacy similar to aripiprazole, suggesting potential efficacy of WS-50030 versus positive and negative symptoms of schizophrenia, comorbid mood symptoms, bipolar disorder, major depressive disorder, and treatment-resistant depression. Furthermore, WS-50030 provides a tool to further explore how combining these mechanisms might differentiate from other antipsychotics or antidepressants.
- Published
- 2009
13. Tetrahydrocarbazole-based serotonin reuptake inhibitor/dopamine D2 partial agonists for the potential treatment of schizophrenia
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Jean Zhang, Julie A. Brennan, Chris G. Kruse, Jan-Hendrik Reinders, Dianne Kowal, Geraldine Ruth Mcfarlane, Feenstra Roelof W, Sara Núñez-García, Andrew C. McCreary, Karen L. Marquis, Radka Graf, Mark H. Pausch, Alexander Greenfield, Margaret Lai, Albert J. Robichaud, Farhana Pruthi, Tikva Carrick, David P. Rotella, Cristina Grosanu, Steven M. Grauer, Rajiah A. Denny, Kelly Sullivan, Rachel Navarra, and Martina A.W. van der Neut
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medicine.medical_treatment ,Serotonin reuptake inhibitor ,Clinical Biochemistry ,Carbazoles ,Pharmaceutical Science ,Pharmacology ,Serotonin 5-HT1 Receptor Antagonists ,Biochemistry ,Partial agonist ,chemistry.chemical_compound ,In vivo ,Dopamine receptor D2 ,Drug Discovery ,medicine ,Animals ,Antipsychotic ,Neurotransmitter ,Molecular Biology ,Receptors, Dopamine D2 ,Organic Chemistry ,Rats ,Disease Models, Animal ,chemistry ,Dopamine Agonists ,Receptor, Serotonin, 5-HT1A ,Schizophrenia ,Molecular Medicine ,Serotonin ,Reuptake inhibitor ,Selective Serotonin Reuptake Inhibitors - Abstract
A 5-fluoro-tetrahydrocarbazole serotonin reuptake inhibitor (SRI) building block was combined with a variety of linkers and dopamine D2 receptor ligands in an attempt to identify potent D2 partial agonist/SRI molecules for treatment of schizophrenia. This approach has the potential to treat a broader range of symptoms compared to existing therapies. Selected compounds in this series demonstrate high affinity for both targets and D2 partial agonism in cell-based and in vivo assays.
- Published
- 2009
14. The orphan GPCR, GPR88, modulates function of the striatal dopamine system: a possible therapeutic target for psychiatric disorders?
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Steven M. Grauer, M Amy Sung, Noel Taylor, Karen L. Marquis, Radka Graf, Lynn Zhang, Feng Liu, Zoë A. Hughes, Mark H. Pausch, Sharon Rosenzweig-Lipson, Janet Paulsen, Nicholas J. Brandon, Sheree F. Logue, Brian Bates, and Virginia L. Pulito
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Male ,medicine.medical_specialty ,Dopamine and cAMP-Regulated Phosphoprotein 32 ,Reflex, Startle ,Apomorphine ,Dopamine ,Striatum ,Biology ,Nucleus accumbens ,Motor Activity ,Neuropsychological Tests ,Receptors, G-Protein-Coupled ,Cellular and Molecular Neuroscience ,Mice ,Neurochemical ,Internal medicine ,Dopamine receptor D2 ,medicine ,Animals ,Humans ,Psychiatry ,Molecular Biology ,Prepulse inhibition ,Mice, Knockout ,Behavior, Animal ,Receptors, Dopamine D2 ,Brain ,Cell Biology ,Risperidone ,Corpus Striatum ,Stereotypy (non-human) ,Endocrinology ,Dopamine Agonists ,Dopamine Antagonists ,Haloperidol ,Female ,medicine.drug ,Antipsychotic Agents - Abstract
In rodents, the orphan G protein-coupled receptor, Gpr88, is highly expressed in brain regions implicated in the pathophysiology of and is modulated by treatments for schizophrenia. We compared striatal function of Gpr88 knockout mice (Gpr88KOs) to wild-type mice using molecular, neurochemical and behavioral tests. Gpr88KOs lacked expression of Gpr88 in striatum, nucleus accumbens and layer IV of cortex. Gpr88KOs had normal striatal dopamine D2 receptor density and affinity and DARPP-32 expression but Gpr88KOs had higher basal striatal phosphorylated DARPP-32 Thr-34. In vivo microdialysis detected lower basal dopamine in Gpr88KOs while amphetamine-induced dopamine release was normal. Behaviorally, Gpr88KOs demonstrated disrupted prepulse inhibition of startle (PPI) and increased sensitivity to apomorphine-induced climbing and stereotypy (AICS) and amphetamine-stimulated locomotor activity. Antipsychotic administration to Gpr88KOs normalized the PPI deficit and blocked AICS. The modulatory role of Gpr88 in striatal dopamine function suggests it may be a new target for treatments for psychiatric disorders.
- Published
- 2009
15. Pharmacological comparison of muscarinic ligands: historical versus more recent muscarinic M1-preferring receptor agonists
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Tikva Carrick, Julia N. Heinrich, John A. Butera, Karen L. Marquis, Scott Christian Mayer, Eugene Tseng, Tim Lock, Shaiu-Ching Sun, Albert J. Uveges, Dianne Kowal, Angela Kramer, Mark H. Pausch, and Michael Popiolek
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Pharmacology ,Agonist ,Receptor, Muscarinic M3 ,medicine.medical_specialty ,Chemistry ,medicine.drug_class ,Receptors, Dopamine D2 ,Receptor, Muscarinic M1 ,Muscarinic acetylcholine receptor M1 ,Muscarinic Agonists ,Talsaclidine ,Sabcomeline ,Ligands ,Cevimeline ,chemistry.chemical_compound ,Endocrinology ,Internal medicine ,Muscarinic acetylcholine receptor ,Receptor, Serotonin, 5-HT2B ,medicine ,Muscarinic acetylcholine receptor M4 ,Xanomeline ,medicine.drug ,Protein Binding - Abstract
In functional assay assessments using the five muscarinic receptor subtypes, a second generation of muscarinic M(1)-preferring receptor agonists [AC-42 (1), AC-260584 (2), 77-LH-28-1 (3) and LY-593039 (4)] was shown to have higher selectivity for muscarinic M(1) over M(3) receptor as compared to historical agonists [talsaclidine (8), sabcomeline (10), xanomeline (11), WAY-132983 (12), cevimeline (9) and NGX-267 (6)]. Another striking difference of these more recent compounds is their affinities for the dopamine D(2) and 5-HT(2B) receptors. Taken together, these results suggest that the newer compounds may have a greater clinical safety profile, especially with regard to muscarinic M(3) receptor-mediated events, than the historical agonists, but their affinities for other receptors may still compromise their use to validate the therapeutic potential of muscarinic M(1) receptor agonists.
- Published
- 2008
16. The identification of neurotensin NTS1 receptor partial agonists through a ligand-based virtual screening approach
- Author
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Michael Popiolek, Margaret Lai, Terrance H. Andree, Mark H. Pausch, Yi Fan, Paul Jeffrey Dollings, and Kelly Sullivan
- Subjects
Agonist ,Models, Molecular ,medicine.drug_class ,Stereochemistry ,Clinical Biochemistry ,Pharmaceutical Science ,CHO Cells ,Ligands ,Biochemistry ,Partial agonist ,chemistry.chemical_compound ,Cricetulus ,Cricetinae ,Drug Discovery ,medicine ,Animals ,Humans ,Receptors, Neurotensin ,Database search engine ,Receptor ,Molecular Biology ,Virtual screening ,Chemistry ,Organic Chemistry ,Antagonist ,Small molecule ,Molecular Medicine ,Neurotensin - Abstract
We identified small molecule NTS1R agonist compounds through virtual screening of the corporate database using a ROCS approach that searches multi-conformer representations efficiently. As a starting point for the ROCS search, we used the known NTS1R selective antagonist, SR-48527, based on the hypothesis that NT agonists and antagonists might share similar binding regions. Conformations were expanded and selected as database search queries based on a cluster analysis. The search provided us with virtual hits that were tested in intracellular calcium mobilization assays of NTS1R agonist and antagonist activities measured in FLIPR format as well as in [ 3 H]NT competition binding studies. The results indicated that two initial hits produced partial agonist activity with potency in the moderate micromolar range.
- Published
- 2008
17. MrgD activation inhibits KCNQ/M-currents and contributes to enhanced neuronal excitability
- Author
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Mark H. Pausch, Seena K. Ajit, Robert A. Crozier, and Edward J. Kaftan
- Subjects
Male ,Patch-Clamp Techniques ,Action Potentials ,Endogeny ,CHO Cells ,KCNQ3 Potassium Channel ,Receptors, G-Protein-Coupled ,Rats, Sprague-Dawley ,Cricetulus ,Dorsal root ganglion ,Cricetinae ,Ganglia, Spinal ,M current ,medicine ,Animals ,KCNQ2 Potassium Channel ,Rats, Long-Evans ,Receptor ,G protein-coupled receptor ,Neurons ,Phospholipase C ,Chemistry ,General Neuroscience ,Articles ,Potassium channel ,Rats ,medicine.anatomical_structure ,nervous system ,Nociceptor ,Neuroscience - Abstract
The recently identified Mas-related gene (Mrg) family of G-protein-coupled receptors is expressed almost exclusively in dorsal root ganglion (DRG) neurons. The expression of one family member, MrgD, is even further confined to IB4+, nonpeptidergic, small-diameter nociceptors. Although the functional consequences of MrgD activation are not known, this expression profile provides intriguing potential for a role in pain sensation or modulation. In a recombinant cell line, we first assessed the functional significance of MrgD activation by coexpressing MrgD with the KCNQ2/3 potassium channel, a channel implicated in pain. Whole-cell voltage-clamp recordings revealed that bath application of the ligand for MrgD, β-alanine, resulted in robust inhibition of KCNQ2/3 activity. Pharmacological blockade of Gi/oand phospholipase C signaling revealed a partial and complete block of the response, respectively. We extended these observations to dissociated DRG neuron cultures by examining MrgD modulation of M-currents (carried primarily by KCNQ2/3). Here too, β-alanine-induced activation of endogenous MrgD inhibited M-currents, but primarily via a pertussis toxin-sensitive pathway. Finally, we assessed the consequence of β-alanine-induced activation of MrgD in phasic neurons. Phasic neurons that fired a single action potential (AP) before β-alanine application fired multiple APs during β-alanine exposure. In sum, we provide evidence for a novel interaction between MrgD and KCNQ/M-type potassium channels that contributes to an increase in excitability of DRG neurons and thus may enhance the signaling of primary afferent nociceptive neurons.
- Published
- 2007
18. Tissue distribution and functional analyses of the constitutively active orphan G protein coupled receptors, GPR26 and GPR78
- Author
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Stanley P. Nawoschik, Eugene Tseng, Kodangattil Sreekumar, Mark H. Pausch, Albert J. Uveges, Lynn Zhang, Philip G. Jones, Brian Bates, Janet E. Paulsen, Jeremy Johnson, and Lan He
- Subjects
Male ,Saccharomyces cerevisiae Proteins ,Arginine ,G protein ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Biophysics ,Mutation, Missense ,Biology ,Biochemistry ,Cell Line ,Receptors, G-Protein-Coupled ,Mice ,Cyclic AMP ,Animals ,Humans ,Receptor ,Molecular Biology ,Peptide sequence ,Endoplasmic Reticulum Chaperone BiP ,G protein-coupled receptor ,Orphan receptor ,chemistry.chemical_classification ,Base Sequence ,Sequence Homology, Amino Acid ,Brain ,GTP-Binding Protein alpha Subunits ,Amino acid ,Glutamine ,chemistry ,Mutagenesis, Site-Directed ,GTP-Binding Protein alpha Subunits, Gq-G11 - Abstract
GPR26 and GPR78 are orphan GPCRs (oGPCRs) that share 51% amino acid sequence identity and are widely expressed in selected tissues of the human brain as well as the developing and adult mouse brain. Investigation of the functional activity of GPR26 and GPR78 via expression in HEK293 cells showed that both proteins are constitutively active and coupled to elevated cAMP production. Accordingly, in yeast, GPR26 demonstrated apparent agonist-independent coupling to a chimeric Gpa1 protein in which the 5 C-terminal amino acids were from Galphas. A comparison of the proteins revealed an atypical glutamine residue in GPR78 in place of the conserved arginine residue (R3.50) in the so-called DRY box. Site-directed mutants R3.50 in GPR26 were constructed and retained their constitutive activity suggesting that these 2 receptors activate G proteins in a manner that is distinct from other group 1 GPCRs.
- Published
- 2006
19. Aplindore (DAB-452), a high affinity selective dopamine D2 receptor partial agonist
- Author
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Geoff Hornby, Margaret Lai, Li-Xin Jiang, Terrance H. Andree, Gary Paul Stack, Julia N. Heinrich, Michael Popiolek, Mark H. Pausch, Julie A. Brennan, Kelly Sullivan, and Karen L. Marquis
- Subjects
Aplindore ,Male ,Indoles ,Quinpirole ,medicine.drug_class ,CHO Cells ,Biology ,Motor Activity ,Partial agonist ,Binding, Competitive ,Rats, Sprague-Dawley ,Radioligand Assay ,Dopamine receptor D1 ,Cricetulus ,Dopamine receptor D2 ,Cricetinae ,Receptors, Adrenergic, alpha-1 ,medicine ,Animals ,Phosphorylation ,Extracellular Signal-Regulated MAP Kinases ,Oxidopamine ,Pharmacology ,Raclopride ,Dose-Response Relationship, Drug ,Receptors, Dopamine D2 ,Dopaminergic ,Receptors, Dopamine D4 ,Receptor antagonist ,Molecular biology ,GTP-Binding Protein alpha Subunits ,Rats ,Substantia Nigra ,Guanosine 5'-O-(3-Thiotriphosphate) ,Dopamine Agonists ,Receptor, Serotonin, 5-HT1A ,Calcium ,Receptors, Serotonin, 5-HT2 ,Endogenous agonist ,medicine.drug - Abstract
The pharmacology of aplindore (DAB-452) was characterized in CHO-K1 cells stably transfected with the human dopamine D(2) receptor short isoform (CHO-D(2s)) and in a behavioral model for post-synaptic agonism in rats. In [(3)H]-spiperone competition binding studies, aplindore showed high affinity for dopamine D(2) and D(3) receptors and low affinity for the dopamine D(4), serotonin (5-HT)(1A), 5-HT(2) receptors and the alpha1-adrenoceptor. The high potency partial agonist activity of aplindore was demonstrated in [(35)S]guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding, extracellular signal-regulated kinase (ERK)-phosphorylation and intracellular calcium flux assay using fluorometric plate reader ([Ca(2+)](i)-FLIPR) format. The [Ca(2+)](i)-FLIPR assay was conducted with CHO-D(2S) receptor cells also stably expressing chimeric G(alphaq/o)-proteins. In all assay modalities, the potencies and intrinsic activities of aplindore were lower than dopamine and higher than aripiprazole. In contrast to the [(35)S]GTPgammaS binding and ERK-phosphorylation assays, the [Ca(2+)](i)-FLIPR assay was able to detect the low partial agonist activity of SDZ 208-912. In unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, aplindore induced contralateral turning, which was blocked by the dopamine D(2) receptor antagonist raclopride. The dopamine D(2) receptor selective partial agonist profile of aplindore suggests that it should be effective for the treatment of dopaminergic-based disorders, such as schizophrenia and Parkinson's disease.
- Published
- 2006
20. Saccharomyces cerevisiae : Applications
- Author
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Donald R. Kirsch, Sanford J. Silverman, and Mark H. Pausch
- Subjects
Genetics ,chemistry.chemical_classification ,Saccharomyces cerevisiae ,Computational biology ,Biology ,biology.organism_classification ,Genome ,Yeast ,law.invention ,Transcriptome ,Enzyme ,chemistry ,law ,Proteome ,Recombinant DNA ,Eukaryotic cell - Abstract
The yeast Saccharomyces cerevisiae has traditionally found useful applications in the production of food and alcoholic beverages. The completed sequence of its genome has revolutionized the use of yeast as a model system for the investigation of eukaryotic cell processes at a whole-genome level. The production of enzymes and recombinant proteins and the development of drug screening assays are commercial applications of yeast cells. Keywords: genome; transcriptome; proteome; two-hybrid screening
- Published
- 2005
21. Functional expression of human and mouse P2Y12 receptors in Saccharomyces cerevisiae
- Author
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Eugene Tseng, Margaret Lai, Mark H. Pausch, Janet E. Paulsen, Brian Bates, and Seung Kwak
- Subjects
Recombinant Fusion Proteins ,Saccharomyces cerevisiae ,Molecular Sequence Data ,Biophysics ,In situ hybridization ,Biochemistry ,Mice ,Genes, Reporter ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Receptor ,Molecular Biology ,In Situ Hybridization ,G protein-coupled receptor ,Reporter gene ,biology ,Receptors, Purinergic P2 ,Brain ,Membrane Proteins ,Cell Biology ,biology.organism_classification ,Fusion protein ,Molecular biology ,Receptors, Purinergic P2Y12 ,Rats ,Gq alpha subunit ,biology.protein ,NIH 3T3 Cells ,GTP-Binding Protein alpha Subunits, Gq-G11 ,Calcium ,Signal transduction ,Sequence Alignment - Abstract
DNA sequences encoding the murine ortholog of the human P2Y12 receptor were cloned. The human and mouse P2Y12 receptors were expressed in a yeast cell-based GPCR expression technology containing chimeric yeast Galpha protein (Gpa1) constructs in which the 5 C-terminal amino acids were identical to corresponding sequences from mammalian Galphai/o proteins. LacZ reporter gene assays of agonist-induced activation of the G protein-coupled mating signal transduction pathway revealed murine P2Y12 functional pharmacological properties that closely resembled those exhibited by the human P2Y12 receptor. In NIH3T3 cells, the mouse P2Y12 stimulated calcium uptake monitored in FLIPR via coupling to a Galphaq/i3 chimeric protein. Murine P2Y12 mRNA was expressed at high levels in the brain and at lower levels in a variety of peripheral tissues. In situ hybridization analysis indicated glia-specific expression within the brain.
- Published
- 2004
22. Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation
- Author
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Beth, Noble, Lorena A, Kallal, Mark H, Pausch, and Jeffrey L, Benovic
- Subjects
G-Protein-Coupled Receptor Kinase 5 ,Insecta ,Time Factors ,Genetic Vectors ,Saccharomyces cerevisiae ,Protein Serine-Threonine Kinases ,Catalysis ,Cell Line ,Animals ,Humans ,Phosphorylation ,Phospholipids ,Dose-Response Relationship, Drug ,Cyclic AMP-Dependent Protein Kinases ,Lipids ,Protein Structure, Tertiary ,Kinetics ,Mutagenesis ,beta-Adrenergic Receptor Kinases ,COS Cells ,Mutation ,Biological Assay ,Receptors, Adrenergic, beta-2 ,Peptides ,Cell Division ,Plasmids ,Protein Binding - Abstract
G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.
- Published
- 2003
23. COMPOUND 1, A POTENT AND SELECTIVE DAO INHIBITOR, DEMONSTRATES EFFICACY IN SEVERAL PRECLINICAL ANIMAL MODELS OF SCHIZOPHRENIA
- Author
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Steven M. Grauer, Rachel Navarra, Julie A. Brennan, Michael Popiolek, John A. Butera, Joseph Zaccardi, Girija Krishnamurthy, Karen L. Marquis, Paul Jeffrey Dollings, Mark H. Pausch, Keith Pitts, and Nicholas J. Brandon
- Subjects
Psychiatry and Mental health ,business.industry ,Animal models of schizophrenia ,Medicine ,Pharmacology ,business ,Biological Psychiatry - Published
- 2010
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