35 results on '"Calver AR"'
Search Results
2. Identification of clinical candidates from the benzazepine class of histamine H3 receptor antagonists.
- Author
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Wilson DM, Apps J, Bailey N, Bamford MJ, Beresford IJ, Brackenborough K, Briggs MA, Brough S, Calver AR, Crook B, Davis RK, Davis RP, Davis S, Dean DK, Harris L, Heslop T, Holland V, Jeffrey P, Panchal TA, Parr CA, Quashie N, Schogger J, Sehmi SS, Stean TO, Steadman JG, Trail B, Wald J, Worby A, Takle AK, Witherington J, and Medhurst AD
- Subjects
- Animals, Benzazepines pharmacokinetics, Dogs, Half-Life, Haplorhini, Histamine H3 Antagonists chemical synthesis, Histamine H3 Antagonists pharmacokinetics, Humans, Male, Microsomes, Liver metabolism, Niacinamide analogs & derivatives, Niacinamide chemistry, Niacinamide pharmacokinetics, Protein Binding, Rats, Rats, Sprague-Dawley, Receptors, Histamine H3 metabolism, Structure-Activity Relationship, Benzazepines chemistry, Histamine H3 Antagonists chemistry, Receptors, Histamine H3 chemistry
- Abstract
This Letter describes the discovery of GSK189254 and GSK239512 that were progressed as clinical candidates to explore the potential of H3 receptor antagonists as novel therapies for the treatment of Alzheimer's disease and other dementias. By carefully controlling the physicochemical properties of the benzazepine series and through the implementation of an aggressive and innovative screening strategy that employed high throughput in vivo assays to efficiently triage compounds, the medicinal chemistry effort was able to rapidly progress the benzazepine class of H3 antagonists through to the identification of clinical candidates with robust in vivo efficacy and excellent developability properties., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
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3. The discovery of the benzazepine class of histamine H3 receptor antagonists.
- Author
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Wilson DM, Apps J, Bailey N, Bamford MJ, Beresford IJ, Briggs MA, Calver AR, Crook B, Davis RP, Davis S, Dean DK, Harris L, Heightman TD, Panchal T, Parr CA, Quashie N, Steadman JG, Schogger J, Sehmi SS, Stean TO, Takle AK, Trail BK, White T, Witherington J, Worby A, and Medhurst AD
- Subjects
- Animals, Benzazepines chemical synthesis, Benzazepines pharmacokinetics, Cytochrome P-450 CYP2D6 chemistry, Cytochrome P-450 CYP2D6 metabolism, Drug Evaluation, Preclinical, Half-Life, Histamine H3 Antagonists chemical synthesis, Histamine H3 Antagonists pharmacokinetics, Humans, Microsomes, Liver metabolism, Protein Binding, Rats, Receptors, Histamine H3 genetics, Receptors, Histamine H3 metabolism, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins genetics, Structure-Activity Relationship, Benzazepines chemistry, Histamine H3 Antagonists chemistry, Receptors, Histamine H3 chemistry
- Abstract
This Letter describes the discovery of a novel series of H3 receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H3 receptor antagonists based on the benzazepine core. Employing an H3 driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
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4. Targeting phosphoinositide 3-kinase δ for the treatment of respiratory diseases.
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Sriskantharajah S, Hamblin N, Worsley S, Calver AR, Hessel EM, and Amour A
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- Asthma enzymology, Epithelial Cells metabolism, Humans, Inflammation metabolism, Isoenzymes antagonists & inhibitors, Isoenzymes metabolism, Macrophages metabolism, Molecular Targeted Therapy, Neutrophils metabolism, Oxidative Stress, Phosphatidylinositol 3-Kinases metabolism, Pulmonary Disease, Chronic Obstructive enzymology, Reactive Oxygen Species metabolism, T-Lymphocytes metabolism, Asthma drug therapy, Phosphoinositide-3 Kinase Inhibitors, Pulmonary Disease, Chronic Obstructive drug therapy
- Abstract
Asthma and chronic obstructive pulmonary disease (COPD) are characterized in their pathogenesis by chronic inflammation in the airways. Phosphoinositide 3-kinase δ (PI3Kδ), a lipid kinase expressed predominantly in leukocytes, is thought to hold much promise as a therapeutic target for such inflammatory conditions. Of particular interest for the treatment of severe respiratory disease is the observation that inhibition of PI3Kδ may restore steroid effectiveness under conditions of oxidative stress. PI3Kδ inhibition may also prevent recruitment of inflammatory cells, including T lymphocytes and neutrophils, as well as the release of proinflammatory mediators, such as cytokines, chemokines, reactive oxygen species, and proteolytic enzymes. In addition, targeting the PI3Kδ pathway could reduce the incidence of pathogen-induced exacerbations by improving macrophage-mediated bacterial clearance. In this review, we discuss the potential and highlight the unknowns of targeting PI3Kδ for the treatment of respiratory disease, focusing on recent developments in the role of the PI3Kδ pathway in inflammatory cell types believed to be critical to the pathogenesis of COPD., (© 2013 New York Academy of Sciences.)
- Published
- 2013
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5. Identification of a novel 5-HT(4) receptor splice variant (r5-HT(4c1)) and preliminary characterisation of specific 5-HT(4a) and 5-HT(4b) receptor antibodies.
- Author
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Ray AM, Kelsell RE, Houp JA, Kelly FM, Medhurst AD, Cox HM, and Calver AR
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- Animals, Antibodies, Monoclonal immunology, Antibodies, Monoclonal metabolism, Base Sequence, Binding, Competitive, Cell Line, Cell Membrane metabolism, Cloning, Molecular, Cyclic AMP metabolism, Female, Gastrointestinal Tract metabolism, Humans, Immunohistochemistry, Male, Molecular Sequence Data, Protein Isoforms, RNA, Messenger genetics, RNA, Messenger metabolism, Rabbits, Radioligand Assay, Rats, Rats, Sprague-Dawley, Receptors, Serotonin, 5-HT4 metabolism, Reverse Transcriptase Polymerase Chain Reaction, Serotonin Receptor Agonists pharmacology, Transfection, Alternative Splicing, Antibodies, Monoclonal pharmacology, Receptors, Serotonin, 5-HT4 genetics
- Abstract
The human 5-hydroxytryptamine (5-HT(4)) receptor is encoded by a highly complex gene which gives rise to at least 10 distinct splice variants. However, the functional relevance of these variants is unknown. In rat, only three such variants have been identified, 5-HT(4a) (r5-HT(4a)), 5-HT(4b) (r5-HT(4b)) and 5-HT(4e) (r5-HT(4e)). In the current study we identify and characterise the pharmacology of a novel rat splice variant (r5-HT(4c1)) and present the first comprehensive analysis of 5-HT(4) splice variant mRNA expression levels throughout the rat gastrointestinal tract. In addition, we describe preliminary characterisation of the first 5-HT(4) splice variant specific antibodies. In transfected cells, r5-HT(4c1) receptor exhibited similar binding properties to r5-HT(4a) and r5-HT(4b). Functional studies showed that 5-HT(4) agonists prucalopride (4-amino-5-chloro-2,3-dihydro-N-[1-(3-methoxypropyl)-4-piperidinyl]-7-benzofuran carboxamide monohydrochloride and renzapride (+/-)-endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3.3.1]non-4-yl)benzamide monohydrochloride) acted as partial agonists at r5-HT(4c1), but full agonists at r5-HT(4a) and r5-HT(4b). Moreover, in contrast to r5-HT(4a) and r5-HT(4b), r5-HT(4c1) was not constitutively active. TaqMan mRNA analysis showed that r5-HT(4a) expression in brain and dorsal root ganglion exceeded that in the gastrointestinal tract, whilst the reverse was true for r5-HT(4b) and r5-HT(4c1). mRNA expression of each variant also increased distally throughout the gastrointestinal tract with the highest levels in the colon. r5-HT(4a) and r5-HT(4b) specific immunoreactivity was abundant on enteric neurons in jejunum, ileum and colon as well as neurons and satellite cells of the dorsal root ganglion. Only r5-HT(4b) immunoreactivity was observed on endocrine cells in the duodenum. These data could have implications in rat models and aid understanding of 5-HT(4) splice variant function.
- Published
- 2009
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6. GABAB receptors role in cell migration and positioning within the ventromedial nucleus of the hypothalamus.
- Author
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McClellan KM, Calver AR, and Tobet SA
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- Animals, Animals, Newborn, Baclofen analogs & derivatives, Baclofen pharmacology, Cell Movement drug effects, Dose-Response Relationship, Drug, Embryo, Mammalian, Estrogen Receptor alpha metabolism, GABA Antagonists pharmacology, In Vitro Techniques, Luminescent Proteins genetics, Mice, Mice, Inbred C57BL, Mice, Transgenic, Neurons drug effects, Protein Subunits metabolism, Receptors, GABA-A metabolism, Receptors, GABA-B deficiency, Ventromedial Hypothalamic Nucleus embryology, Ventromedial Hypothalamic Nucleus growth & development, Cell Movement physiology, Neurons physiology, Receptors, GABA-B physiology, Ventromedial Hypothalamic Nucleus cytology
- Abstract
The ventromedial (VMN) and arcuate (ARC) nuclei of the hypothalamus are bilateral nuclear groups at the base of the hypothalamus that are organized through the aggregation of neurons born along the third ventricle that migrate laterally. During development, GABAergic neurons and fibers surround the forming (or primordial) VMN while neurons containing GABA receptors are found within the boundaries of the emerging nucleus. To investigate the role that GABAB receptors play in establishing the VMN, Thy-1 yellow fluorescent protein (YFP) mice were utilized for live video microscopy studies. The Thy-1 promoter drives YFP expression in regions of the hypothalamus during development. Administration of the GABAB receptor antagonist saclofen and the GABAA receptor antagonist bicuculline selectively increased the rate of VMN cell movement in slices placed in vitro at embryonic day 14, when cells that form both the ARC and VMN are migrating away from the proliferative zone surrounding the third ventricle. To further test the role of GABAB receptors in VMN development, GABAB receptor knockout mice were used to examine changes in the positions of phenotypically identified cells within the VMN. Cells containing immunoreactive estrogen receptors (ER) alpha were located in the ventrolateral quadrant of the wild type VMN. In GABABR1 knockout mice, these ERalpha positive neurons were located in more dorsal positions at postnatal day (P) 0 and P4. We conclude that GABA alters cell migration and its effect on final cell positioning may lead to changes in the circuitry and connections within specific nuclei of the developing hypothalamus.
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- 2008
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7. GSK189254, a novel H3 receptor antagonist that binds to histamine H3 receptors in Alzheimer's disease brain and improves cognitive performance in preclinical models.
- Author
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Medhurst AD, Atkins AR, Beresford IJ, Brackenborough K, Briggs MA, Calver AR, Cilia J, Cluderay JE, Crook B, Davis JB, Davis RK, Davis RP, Dawson LA, Foley AG, Gartlon J, Gonzalez MI, Heslop T, Hirst WD, Jennings C, Jones DN, Lacroix LP, Martyn A, Ociepka S, Ray A, Regan CM, Roberts JC, Schogger J, Southam E, Stean TO, Trail BK, Upton N, Wadsworth G, Wald JA, White T, Witherington J, Woolley ML, Worby A, and Wilson DM
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Alzheimer Disease metabolism, Alzheimer Disease pathology, Alzheimer Disease physiopathology, Animals, Benzazepines metabolism, Benzazepines pharmacokinetics, Binding, Competitive, Brain metabolism, Brain pathology, Cell Line, Dogs, Histamine Agonists metabolism, Histamine Agonists pharmacokinetics, Histamine Agonists pharmacology, Histamine Antagonists metabolism, Histamine Antagonists pharmacokinetics, Humans, Male, Maze Learning drug effects, Mice, Middle Aged, Neurotransmitter Agents metabolism, Niacinamide metabolism, Niacinamide pharmacokinetics, Niacinamide pharmacology, Nootropic Agents metabolism, Nootropic Agents pharmacokinetics, Rats, Rats, Sprague-Dawley, Rats, Wistar, Receptors, Histamine H3 analysis, Sus scrofa, Benzazepines pharmacology, Brain drug effects, Histamine Antagonists pharmacology, Niacinamide analogs & derivatives, Nootropic Agents pharmacology, Receptors, Histamine H3 metabolism
- Abstract
6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H(3) receptor antagonist with high affinity for human (pK(i) = 9.59 -9.90) and rat (pK(i) = 8.51-9.17) H(3) receptors. GSK189254 is >10,000-fold selective for human H(3) receptors versus other targets tested, and it exhibited potent functional antagonism (pA(2) = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC(50) = 8.20 versus basal guanosine 5'-O-(3-[(35)S]thio)triphosphate binding] at the human recombinant H(3) receptor. In vitro autoradiography demonstrated specific [(3)H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H(3) binding was detected in medial temporal cortex samples from severe cases of Alzheimer's disease, suggesting for the first time that H(3) receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(-)-alpha-methyl[imidazole-2,5(n)-(3)H]histamine dihydrochloride ([(3)H]R-alpha-methylhistamine) binding (ED(50) = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3-3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50) = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimer's disease and other cognitive disorders.
- Published
- 2007
- Full Text
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8. Structurally novel histamine H3 receptor antagonists GSK207040 and GSK334429 improve scopolamine-induced memory impairment and capsaicin-induced secondary allodynia in rats.
- Author
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Medhurst AD, Briggs MA, Bruton G, Calver AR, Chessell I, Crook B, Davis JB, Davis RP, Foley AG, Heslop T, Hirst WD, Medhurst SJ, Ociepka S, Ray A, Regan CM, Sargent B, Schogger J, Stean TO, Trail BK, Upton N, White T, Orlek B, and Wilson DM
- Subjects
- Analgesics pharmacokinetics, Analgesics pharmacology, Analgesics therapeutic use, Animals, Avoidance Learning drug effects, Azepines administration & dosage, Azepines pharmacokinetics, Benzazepines pharmacokinetics, Benzazepines pharmacology, Central Nervous System drug effects, Drinking drug effects, Histamine Agonists pharmacokinetics, Histamine Agonists pharmacology, Histamine Antagonists pharmacokinetics, Histamine Antagonists pharmacology, Humans, Male, Memory Disorders chemically induced, Neuralgia chemically induced, Pyrazines pharmacokinetics, Pyrazines pharmacology, Pyridines administration & dosage, Pyridines pharmacokinetics, Rats, Rats, Sprague-Dawley, Azepines therapeutic use, Benzazepines therapeutic use, Capsaicin, Histamine Antagonists therapeutic use, Memory Disorders drug therapy, Neuralgia drug therapy, Pyrazines therapeutic use, Pyridines therapeutic use, Receptors, Histamine H3 metabolism, Scopolamine
- Abstract
GSK207040 (5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-2-pyrazinecarboxamide) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1H-1,4-diazepine) are novel and selective non-imidazole histamine H(3) receptor antagonists from distinct chemical series with high affinity for human (pK(i)=9.67+/-0.06 and 9.49+/-0.09, respectively) and rat (pK(i)=9.08+/-0.16 and 9.12+/-0.14, respectively) H(3) receptors expressed in cerebral cortex. At the human recombinant H(3) receptor, GSK207040 and GSK334429 were potent functional antagonists (pA(2)=9.26+/-0.04 and 8.84+/-0.04, respectively versus H(3) agonist-induced changes in cAMP) and exhibited inverse agonist properties (pIC(50)=9.20+/-0.36 and 8.59+/-0.04 versus basal GTPgammaS binding). Following oral administration, GSK207040 and GSK334429 potently inhibited cortical ex vivo [(3)H]-R-alpha-methylhistamine binding (ED(50)=0.03 and 0.35 mg/kg, respectively). Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50)=0.02 and 0.11 mg/kg p.o. for GSK207040 and GSK334429, respectively). In more pathophysiologically relevant pharmacodynamic models, GSK207040 (0.1, 0.3, 1 and 3mg/kg p.o.) and GSK334429 (0.3, 1 and 3mg/kg p.o.) significantly reversed amnesia induced by the cholinergic antagonist scopolamine in a passive avoidance paradigm. In addition, GSK207040 (0.1, 0.3 and 1mg/kg p.o.) and GSK334429 (3 and 10mg/kg p.o.) significantly reversed capsaicin-induced reductions in paw withdrawal threshold, suggesting for the first time that blockade of H(3) receptors may be able to reduce tactile allodynia. Novel H(3) receptor antagonists such as GSK207040 and GSK334429 may therefore have therapeutic potential not only in dementia but also in neuropathic pain.
- Published
- 2007
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9. Dissociation and trafficking of rat GABAB receptor heterodimer upon chronic capsaicin stimulation.
- Author
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Laffray S, Tan K, Dulluc J, Bouali-Benazzouz R, Calver AR, Nagy F, and Landry M
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- Animals, Animals, Newborn, Bicuculline pharmacology, Coculture Techniques methods, Dose-Response Relationship, Drug, Drug Interactions, Endocytosis drug effects, Endocytosis physiology, GABA Antagonists pharmacology, Ganglia, Spinal cytology, Ganglia, Spinal physiology, Ionophores pharmacology, Monensin pharmacology, Nerve Tissue Proteins metabolism, Neurons, Afferent physiology, Organ Culture Techniques, Protein Transport drug effects, Rats, Spinal Cord cytology, Spinal Cord physiology, Time Factors, Transfection methods, Capsaicin pharmacology, Neurons, Afferent drug effects, Protein Subunits metabolism, Receptors, GABA-B metabolism
- Abstract
Gamma-aminobutyric acid type B receptors (GABAB) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABAB1 and GABAB2 subunits, a process that occurs within the endoplasmic reticulum. However, the mechanisms that regulate GABAB receptor oligomerization at the plasma membrane remain largely unknown. We first characterized the functional cytoarchitecture of an organotypic co-culture model of rat dorsal root ganglia and spinal cord. Subsequently, we studied the interactions between GABAB subunits after chronic stimulation of sensory fibres with capsaicin. Surface labelling of recombinant proteins showed a decrease in subunit co-localization and GABAB2 labelling, after capsaicin treatment. In these conditions, fluorescence lifetime imaging measurements further demonstrated a loss of interactions between green fluorescent protein-GABAB1b and t-dimer discosoma sp red fluorescent protein-GABAB2 subunits. Finally, we established that the GABAB receptor undergoes clathrin-dependent internalization and rapid recycling to the plasma membrane following activation with baclofen, a GABAB agonist. However, in cultures chronically stimulated with capsaicin, the agonist-induced endocytosis was decreased, reflecting changes in the dimeric state of the receptor. Taken together, our results indicate that the chronic stimulation of sensory fibres can dissociate the GABAB heterodimer and alters its responsiveness to the endogenous ligand. Chronic stimulation thus modulates receptor oligomerization, providing additional levels of control of signalling.
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- 2007
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10. Mechanisms contributing to the exacerbated epileptiform activity in hippocampal slices expressing a C-terminal truncated GABA(B2) receptor subunit.
- Author
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Thuault SJ, Brown JT, Calver AR, Collingridge GL, Randall A, and Davies CH
- Subjects
- 4-Aminopyridine pharmacology, Animals, Bicuculline pharmacology, Drug Interactions, Electric Stimulation methods, Embryo, Mammalian, Epilepsy genetics, Excitatory Amino Acid Antagonists pharmacology, Excitatory Postsynaptic Potentials drug effects, GABA Antagonists pharmacology, Gene Deletion, In Vitro Techniques, Magnesium metabolism, Membrane Potentials drug effects, Membrane Potentials physiology, Mice, Mice, Knockout, Phosphinic Acids pharmacology, Potassium Channel Blockers pharmacology, Propanolamines pharmacology, Pyramidal Cells, Quinolinium Compounds pharmacology, Receptors, GABA-B chemistry, Receptors, GABA-B deficiency, Time Factors, Valine analogs & derivatives, Valine pharmacology, Epilepsy metabolism, Excitatory Postsynaptic Potentials physiology, Hippocampus metabolism, Receptors, GABA-B metabolism
- Abstract
GABAergic synaptic transmission plays an important role in the patterning of epileptiform activity. We have previously shown that global loss of GABA(B) receptor function due to transgenic deletion of the GABA(B1) receptor subunit exacerbates epileptiform activity induced by pharmacological manipulations in hippocampal slices. Here we show that a similar hyperexcitable phenotype is observed in hippocampal slices prepared from a transgenic mouse expressing a GABA(B2) receptor subunit lacking its C terminal tail (the DeltaGB2-Ct mouse); a molecular manipulation that also produces complete loss of GABA(B) receptor function. Thus, epileptiform bursts that are sensitive to NMDA receptor antagonists (induced by either the GABA(A) receptor antagonist bicuculline (10muM) or removal of extracellular Mg(2+)) were significantly longer in duration in DeltaGB2-Ct slices relative to WT slices. We now extend these observations to demonstrate that a stimulus train induced bursting (STIB) protocol also evokes significantly longer bicuculline sensitive bursts of activity in DeltaGB2-Ct slices compared to WT. Furthermore, synchronous GABA(A) receptor-mediated potentials recorded in the presence of the potassium channel blocker 4-aminopyridine (4-AP, 100muM) and the ionotropic glutamate receptor antagonists NBQX (20muM) and D-AP5 (50muM) were significantly prolonged in duration in DeltaGB2-Ct versus WT slices. These data suggest that the loss of GABA(B) receptor function in DeltaGB2-Ct hippocampal slices promotes depolarising GABA(A) receptor-mediated events, which in turn, leads to the generation of ictal-like events, which may contribute to the epilepsy phenotype observed in vivo.
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- 2005
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11. Multiple motifs regulate the trafficking of GABA(B) receptors at distinct checkpoints within the secretory pathway.
- Author
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Restituito S, Couve A, Bawagan H, Jourdain S, Pangalos MN, Calver AR, Freeman KB, and Moss SJ
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- Amino Acid Motifs physiology, Amino Acid Sequence, Animals, COS Cells, Cells, Cultured, Chlorocebus aethiops, Molecular Sequence Data, Protein Transport physiology, Rats, Receptors, GABA-B genetics, Signal Transduction physiology, Hippocampus metabolism, Receptors, GABA-B metabolism
- Abstract
gamma-Aminobutyric acid type B receptors (GABA(B)) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABA(B)R1 and GABA(B)R2 subunits, a process that occurs within the endoplasmic reticulum (ER). However, the mechanisms that regulate receptor surface expression remain largely unknown. Here, we demonstrate that access to the cell surface for GABA(B)R1 is sequentially controlled by an RSR(R) motif and a LL motif within its cytoplasmic domain. In addition, we reveal that msec7-1, a guanine-nucleotide-exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of GTPases, critical regulators of vesicular membrane trafficking, interacts with GABA(B)R1 via the LL motif in this subunit. Finally, we establish that msec7-1 modulates the cell surface expression of GABA(B) receptors, a process that is dependent upon the integrity of the LL motif in GABA(B)R1. Together, our results demonstrate that the cell surface expression of the GABA(B)R1 subunit is regulated by multiple motifs, which act at distinct checkpoints in the secretory pathway, and also suggest a novel role for msec7-1 in regulating the membrane trafficking of GABA(B)R1 subunits.
- Published
- 2005
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12. Edg8/S1P5: an oligodendroglial receptor with dual function on process retraction and cell survival.
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Jaillard C, Harrison S, Stankoff B, Aigrot MS, Calver AR, Duddy G, Walsh FS, Pangalos MN, Arimura N, Kaibuchi K, Zalc B, and Lubetzki C
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- Amino Acid Sequence, Animals, Ankyrins analysis, Brain cytology, Brain growth & development, Brain Chemistry, Cell Differentiation, Cell Lineage, Cell Shape drug effects, Cell Surface Extensions drug effects, Cell Survival drug effects, Cells, Cultured drug effects, Cells, Cultured metabolism, Cells, Cultured ultrastructure, Crosses, Genetic, Female, GTP-Binding Protein alpha Subunit, Gi2, GTP-Binding Protein alpha Subunits, Gi-Go physiology, Intercellular Signaling Peptides and Proteins, Intracellular Signaling Peptides and Proteins, Kv1.1 Potassium Channel, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Molecular Sequence Data, Nerve Tissue Proteins deficiency, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Oligodendroglia drug effects, Oligodendroglia ultrastructure, Phosphorylation, Potassium Channels, Voltage-Gated analysis, Protein Processing, Post-Translational, Protein Serine-Threonine Kinases metabolism, Protein Serine-Threonine Kinases physiology, Proto-Oncogene Proteins physiology, Proto-Oncogene Proteins c-akt, RNA, Messenger analysis, RNA, Small Interfering pharmacology, Rats, Rats, Wistar, Receptors, Lysosphingolipid deficiency, Receptors, Lysosphingolipid genetics, Signal Transduction drug effects, Signal Transduction physiology, Sphingosine pharmacology, rho-Associated Kinases, Cell Surface Extensions physiology, Lysophospholipids pharmacology, Nerve Tissue Proteins physiology, Oligodendroglia metabolism, Receptors, Lysosphingolipid physiology, Sphingosine analogs & derivatives
- Abstract
Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: sphingosine-1-phosphate (S1P) or lysophosphatidic acid. We show that in the CNS, expression of Edg8/S1P5, a high-affinity S1P receptor, is restricted to oligodendrocytes and expressed throughout development from the immature stages to the mature myelin-forming cell. S1P activation of Edg8/S1P5 on O4-positive pre-oligodendrocytes induced process retraction via a Rho kinase/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S1P on these cells derived from Edg8/S1P5-deficient mice. Edg8/S1P5-mediated process retraction was restricted to immature cells and was no longer observed at later developmental stages. In contrast, S1P activation promoted the survival of mature oligodendrocytes but not of pre-oligodendrocytes. The S1P-induced survival of mature oligodendrocytes was mediated through a pertussis toxin-sensitive, Akt-dependent pathway. Our data demonstrate that Edg8/S1P5 activation on oligodendroglial cells modulates two distinct functional pathways mediating either process retraction or cell survival and that these effects depend on the developmental stage of the cell.
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- 2005
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13. Gamma-hydroxybutyric acid (GHB) and gamma-aminobutyric acidB receptor (GABABR) binding sites are distinctive from one another: molecular evidence.
- Author
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Wu Y, Ali S, Ahmadian G, Liu CC, Wang YT, Gibson KM, Calver AR, Francis J, Pangalos MN, and Carter Snead O 3rd
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- Animals, Binding Sites physiology, Brain metabolism, Cell Line, Dose-Response Relationship, Drug, Humans, Mice, Mice, Knockout, Receptors, GABA-B deficiency, Sodium Oxybate metabolism, Receptors, Cell Surface genetics, Receptors, Cell Surface metabolism, Receptors, GABA-B genetics, Receptors, GABA-B metabolism
- Abstract
gamma-Hydroxybutyric Acid (GHB) is thought to be a weak partial agonist at the gamma-aminobutyric acid(B) Receptor (GABA(B)R), but the precise relationship of the GHB receptor (GHBR) to the GABA(B)R remains unclear. In order to test the hypothesis that the GHBR is not identical to the GABA(B)R, we conducted two groups of experiments. First, GABA(B)R subtype 1 (R1) and/or subtype 2 (R2) were over expressed in HEK 293 cells and membrane binding studies on the transfected cells done using [(3)H]GHB and [(3)H] (2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid ([(3)H]NCS-382). The latter is a specific antagonist at the GHB binding site. Second, [(3)H]GHB and [(3)H]NCS-382 autoradiographic binding studies were done on the brains of mice in which the gene for GABA(B)R1a was deleted. Such mice do not have a functioning GABA(B)R. There was no detectable specific [(3)H]GHB or [(3)H]NCS-382 binding in HEK 293 cells transfected with GABA(B)R1, R2, or R1/R2. Binding to [(3)H]CGP54626A, a high affinity GABA(B)R antagonist, was absent in GABA(B)R1a(-/-) mice. There was no difference in [(3)H]NCS-382 binding observed in the brains of GABA(B)R1a(-/-), GABA(B)R1a(+/-) or GABA(B)R1a(+/+) mice. Specific [(3)H]GHB binding was observed in the brain of GABA(B)R1a(-/-) mice but was significantly lower than in wild type mice. These data support the hypothesis that the GHB binding site is separate and distinct from the GABA(B)R.
- Published
- 2004
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14. The GABA(B2) subunit is critical for the trafficking and function of native GABA(B) receptors.
- Author
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Thuault SJ, Brown JT, Sheardown SA, Jourdain S, Fairfax B, Spencer JP, Restituito S, Nation JH, Topps S, Medhurst AD, Randall AD, Couve A, Moss SJ, Collingridge GL, Pangalos MN, Davies CH, and Calver AR
- Subjects
- Animals, Brain, Corpus Striatum metabolism, Gene Deletion, Mice, Mutation, Phenotype, Protein Transport, Receptors, GABA-B immunology, Receptors, GABA-B metabolism, Receptors, GABA-B genetics, Receptors, Presynaptic metabolism, Synapses metabolism
- Abstract
Studies in heterologous systems have demonstrated that heterodimerisation of the two GABA(B) receptor subunits appears to be crucial for the trafficking and signalling of the receptor. Gene targeting of the GABA(B1) gene has demonstrated that the expression of GABA(B1) is essential for GABA(B) receptor function in the central nervous system (CNS). However, the contribution of the GABA(B2) subunit in the formation of native GABA(B) receptors is still unclear, in particular whether other proteins can substitute for this subunit. We have created a transgenic mouse in which the endogenous GABA(B2) gene has been mutated in order to express a C-terminally truncated version of the protein. As a result, the GABA(B1) subunit does not reach the cell surface and concomitantly both pre- and post-synaptic GABA(B) receptor functions are abolished. Taken together with previous gene deletion studies for the GABA(B1) subunit, this suggests that classical GABA(B) function in the brain is exclusively mediated by GABA(B1/2) heteromers.
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- 2004
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15. Unravelling the unusual signalling properties of the GABA(B) receptor.
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Couve A, Calver AR, Fairfax B, Moss SJ, and Pangalos MN
- Subjects
- Animals, Cell Membrane metabolism, Central Nervous System metabolism, Dimerization, Humans, Mice, Mice, Transgenic, Phosphorylation, Receptors, GABA-B metabolism, Receptors, GABA-B physiology, Signal Transduction physiology
- Abstract
GABA(B) receptors are the cornerstone receptors in the modulation of inhibitory signalling in the central nervous system and continue to be targets for the amelioration of a number of neuropsychiatric and neurological disorders. Unravelling the molecular identity of this receptor has spurred much research over the past five or so years and generated a renewed interest and excitement in the field. Many questions are being answered and lessons learnt, not only about GABA(B) receptor function but also about general mechanisms of G-protein-coupled receptor signalling. However, as questions are being answered as many new questions are being raised and many GABA(B)-related conundrums continue to remain unanswered. In this report, we review some of the most recent work in the area of GABA(B) receptor research. In particular, we focus our attentions on the emerging mechanisms thought to be important in GABA(B) receptor signalling and the growing complex of associated proteins that we consider to be part of the GABA(B) receptor "signalosome."
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- 2004
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16. Marlin-1, a novel RNA-binding protein associates with GABA receptors.
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Couve A, Restituito S, Brandon JM, Charles KJ, Bawagan H, Freeman KB, Pangalos MN, Calver AR, and Moss SJ
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- Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, COS Cells, Hippocampus cytology, Humans, Mice, Molecular Sequence Data, Neurons cytology, RNA, Messenger metabolism, Rats, Sequence Homology, Amino Acid, Superior Cervical Ganglion cytology, Synaptic Transmission physiology, Neurons metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Receptors, GABA-B metabolism
- Abstract
GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission.
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- 2004
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17. Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability.
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Fairfax BP, Pitcher JA, Scott MG, Calver AR, Pangalos MN, Moss SJ, and Couve A
- Subjects
- Animals, Arrestin metabolism, Baclofen pharmacology, Biotinylation, COS Cells, Calcium metabolism, Cell Line, Cells, Cultured, Cerebral Cortex cytology, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, DNA, Complementary metabolism, Dimerization, Endocytosis, Enzyme Activation, GABA Agonists pharmacology, GABA-B Receptor Agonists, Hippocampus cytology, Humans, Microscopy, Fluorescence, Neurons metabolism, Phosphorylation, Plasmids metabolism, Precipitin Tests, Protein Binding, Rats, Receptors, Adrenergic, beta metabolism, Receptors, GABA-B metabolism, Temperature, Time Factors, Cell Membrane metabolism, Receptors, GABA-B chemistry
- Abstract
GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.
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- 2004
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18. Modulation of hippocampal excitability by 5-HT4 receptor agonists persists in a transgenic model of Alzheimer's disease.
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Spencer JP, Brown JT, Richardson JC, Medhurst AD, Sehmi SS, Calver AR, and Randall AD
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- Alzheimer Disease metabolism, Amyloid beta-Peptides metabolism, Animals, Animals, Genetically Modified, Benzofurans pharmacology, Dioxanes pharmacology, Disease Models, Animal, Excitatory Postsynaptic Potentials physiology, Hippocampus drug effects, Humans, Indoles pharmacology, Male, Neurons drug effects, Organ Culture Techniques, Oxadiazoles pharmacology, Patch-Clamp Techniques, Rats, Receptors, Serotonin, 5-HT4 metabolism, Serotonin Antagonists pharmacology, Sulfonamides pharmacology, Alzheimer Disease physiopathology, Amyloid beta-Peptides genetics, Hippocampus metabolism, Neurons metabolism, Serotonin Receptor Agonists pharmacology
- Abstract
5-HT(4) receptors are widely distributed in both peripheral and central nervous systems where they couple, via a G-protein, to the activation of adenylate cyclase. In the brain, the highest 5-HT(4) receptor densities are found in the limbic system, including the hippocampus and frontal cortex. It has been suggested that activation of these receptors may be of therapeutic benefit in diseases that produce cognitive deficits such as Alzheimer's disease (AD). Previous electrophysiological studies have shown that the 5-HT(4) agonist, Zacopride, can increase population spike amplitude recorded in region CA1 of rat hippocampal slices in a cyclic AMP (cAMP)/cAMP-dependent protein kinase A-dependent manner. We report here that the 5-HT(4) agonist, Prucalopride, and the 5-HT(4) partial agonist, SL65.0155, produce a similar effect in rat hippocampal slices and that the specific 5-HT(4) antagonist, GR113808, blocks these effects. To investigate the potential use of 5-HT(4) agonists in the treatment of AD, Prucalopride was applied to hippocampal slices from a transgenic mouse line that overexpresses the Abeta peptide. Despite the deficit in synaptic transmission present in these mice, the percentage increase of the CA1 population spike induced by Prucalopride was the same as that observed in wild-type mice. These data support 5-HT(4) receptors as a target for cognitive enhancement and suggest that a partial agonist would be sufficient to produce benefits, while reducing potential peripheral side effects. In addition, we show that 5-HT(4) receptors remain functional in the presence of excess Abeta peptide and may therefore be a useful target in AD.
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- 2004
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19. Distribution of a GABAB-like receptor protein in the rat central nervous system.
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Charles KJ, Calver AR, Jourdain S, and Pangalos MN
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- Animals, Astrocytes metabolism, Calbindin 2, Central Nervous System anatomy & histology, Computational Biology methods, Glial Fibrillary Acidic Protein metabolism, Immunohistochemistry, Male, Neurons metabolism, Parvalbumins metabolism, Peptides immunology, Rabbits, Rats, Rats, Sprague-Dawley, S100 Calcium Binding Protein G metabolism, Sequence Homology, Amino Acid, Central Nervous System metabolism, Receptors, GABA-B metabolism
- Abstract
Using a homology-based bioinformatics approach we have identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) may be a putative novel GABA(B) receptor subunit. We have developed a rabbit polyclonal antisera specific to the GABA(BL) protein and assessed the distribution of GABA(BL) in the rat CNS by immunohistochemistry. Protein expression was particularly dense in regions previously shown to contain known GABA(B) receptor subunits. Dense immunoreactivity was observed in the cortex, major subfields of the hippocampus and the dentate gyrus. GABA(BL) labelling was very conspicuous in the cerebellum, both in the granule cell layer and in Purkinje cells, and was also observed in the substantia gelatinosa and ventral horn motor neurons of the spinal cord. GABA(BL) immunoreactivity was also noted in a subset of parvalbumin positive hippocampal interneurons. Our data suggest a widespread distribution of GABA(BL) throughout the rat CNS.
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- 2003
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20. Molecular cloning and characterisation of a novel GABAB-related G-protein coupled receptor.
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Calver AR, Michalovich D, Testa TT, Robbins MJ, Jaillard C, Hill J, Szekeres PG, Charles KJ, Jourdain S, Holbrook JD, Boyfield I, Patel N, Medhurst AD, and Pangalos MN
- Subjects
- Amino Acid Sequence genetics, Animals, Base Sequence genetics, Cells, Cultured, Chromosome Mapping, Chromosomes, Human, Pair 3 genetics, Cloning, Molecular, DNA, Complementary analysis, DNA, Complementary genetics, GTP-Binding Proteins genetics, Humans, Immunohistochemistry, Male, Mice, Molecular Sequence Data, Molecular Structure, Phylogeny, Protein Structure, Tertiary genetics, Protein Subunits genetics, Rats, Receptors, GABA-B genetics, Brain metabolism, GTP-Binding Proteins isolation & purification, Protein Subunits isolation & purification, Receptors, GABA-B isolation & purification
- Abstract
Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.
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- 2003
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21. mRNA distribution analysis of human TRPC family in CNS and peripheral tissues.
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Riccio A, Medhurst AD, Mattei C, Kelsell RE, Calver AR, Randall AD, Benham CD, and Pangalos MN
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- Animals, Calcium Channels genetics, Cell Line, Female, Gene Expression Profiling, Humans, Male, Molecular Sequence Data, TRPC Cation Channels, Tissue Distribution, Calcium Channels metabolism, Central Nervous System metabolism, RNA, Messenger metabolism
- Abstract
The mammalian homologues of the Drosophila transient receptor potential (TRP) channel are plasma membrane proteins involved in the regulation of cellular Ca(2+) influx. These ion channels can be activated subsequent to either depletion of Ca(2+) from internal stores or through receptor-mediated processes. The mRNA expression patterns of several individual mammalian short transient receptor potential channels (TRPCs) have been described. Cross-comparisons between these data, however, are at best difficult predominantly due to the non-quantitative methods used. Furthermore there is limited data on the expression of TRPC family members in human tissues. In the present study we used a single technique, namely TaqMan real-time quantitative RT-PCR, to investigate the mRNA distribution of human TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 (hTRPCs) in discrete human brain areas, peripheral tissues as well as a panel of cell-lines. All hTRPCs studied were widely expressed within CNS and significant peripheral expression was often observed. Despite this, each channel exhibited a distinctive hallmark distribution profile. hTRPC1 was widely expressed in CNS and peripheral tissues, whereas hTRPC3 and hTRPC5 were predominantly expressed in tissues of CNS. hTRPC4 mRNA was detected in CNS and certain peripheral tissues such as bone, heart and prostate. hTRPC6 was homogeneously expressed throughout the CNS and peripheral tissues with the highest levels in placenta and lung. hTRPC7 mRNA was also broadly expressed in CNS as well as some peripheral tissues. The pattern of expression of the TRPCs was quite different in the various cell lines examined. TRPC3 and TRPC6 were selectively present in HEK-293 cells whilst TRPC1 was broadly distributed in the cell lines analyzed. In contrast TRPC4 and TRPC5 mRNAs were predominantly expressed in HK-2 and HEK-293 cell lines respectively. TRPC7 was selectively expressed in COS-1, COS-7 and HK-2 cell lines. These results show tissue- and cell-specific co-expression of multiple TRPC forms indicating widespread potential for formation of heteromeric channels. These data will be useful in the complex task of relating channel subunit composition to function in native cells.
- Published
- 2002
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22. GABA(B) receptors: from monogamy to promiscuity.
- Author
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Calver AR, Davies CH, and Pangalos M
- Subjects
- Animals, Humans, Ligands, Neural Inhibition drug effects, Neural Inhibition genetics, Protein Structure, Tertiary drug effects, Protein Structure, Tertiary genetics, Protein Subunits drug effects, Protein Subunits genetics, Protein Subunits metabolism, Receptors, GABA-B drug effects, Receptors, GABA-B genetics, Synapses drug effects, Synaptic Transmission drug effects, Receptors, GABA-B deficiency, Synapses metabolism, Synaptic Transmission genetics
- Abstract
The aim of this review is firstly to describe the current understanding of the diverse physiology and pharmacology of GABA(B) receptors in vivo. We will then focus on recent advances made, since the identification of the GABA(B) receptor subunit genes, in our knowledge of the molecular nature of the receptor, and the recently discovered molecular determinants of functions such as ligand binding, trafficking and signalling. We will conclude with a summary of the GABA(B) receptor-interacting proteins that have been described thus far, and discuss how these may, at least in part, account for the paradox of varied receptor pharmacology in the potential context of a single heterodimeric GABA(B) receptor., (Copyright 2003 S. Karger AG, Basel)
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- 2002
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23. Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.
- Author
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Couve A, Thomas P, Calver AR, Hirst WD, Pangalos MN, Walsh FS, Smart TG, and Moss SJ
- Subjects
- Animals, Brain metabolism, Brain Chemistry, CHO Cells, COS Cells, Cell Membrane chemistry, Cell Membrane metabolism, Cells, Cultured, Cricetinae, Cyclic AMP metabolism, Cyclic AMP pharmacology, Cyclic AMP-Dependent Protein Kinases antagonists & inhibitors, Enzyme Inhibitors pharmacology, GABA Agonists pharmacology, GABA-B Receptor Agonists, Humans, Neurons cytology, Neurons drug effects, Neurons metabolism, Patch-Clamp Techniques, Phosphoproteins agonists, Phosphoproteins analysis, Phosphoproteins metabolism, Phosphorylation, Potassium Channels metabolism, Protein Isoforms agonists, Protein Isoforms analysis, Protein Isoforms metabolism, Rats, Receptors, GABA-B analysis, Recombinant Proteins metabolism, Signal Transduction physiology, Cyclic AMP-Dependent Protein Kinases metabolism, Receptors, GABA-B metabolism
- Abstract
GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.
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- 2002
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24. Cloning and functional expression of human short TRP7, a candidate protein for store-operated Ca2+ influx.
- Author
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Riccio A, Mattei C, Kelsell RE, Medhurst AD, Calver AR, Randall AD, Davis JB, Benham CD, and Pangalos MN
- Subjects
- Amino Acid Sequence, Brain metabolism, Calcium Channel Blockers pharmacology, Cell Line, Central Nervous System embryology, Cloning, Molecular, DNA, Complementary metabolism, Enzyme Inhibitors pharmacology, Epitopes, Exons, Female, Gene Library, Humans, Imidazoles pharmacology, Ion Channels metabolism, Kidney metabolism, Male, Manganese metabolism, Molecular Sequence Data, Oligonucleotides, Antisense pharmacology, Phylogeny, Pituitary Gland metabolism, Protein Binding, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, TRPM Cation Channels, Thapsigargin pharmacology, Time Factors, Tissue Distribution, Transfection, Calcium metabolism, Ion Channels chemistry, Membrane Proteins
- Abstract
The regulation and control of plasma membrane Ca(2+) fluxes is critical for the initiation and maintenance of a variety of signal transduction cascades. Recently, the study of transient receptor potential channels (TRPs) has suggested that these proteins have an important role to play in mediating capacitative calcium entry. In this study, we have isolated a cDNA from human brain that encodes a novel transient receptor potential channel termed human TRP7 (hTRP7). hTRP7 is a member of the short TRP channel family and is 98% homologous to mouse TRP7 (mTRP7). At the mRNA level hTRP7 was widely expressed in tissues of the central nervous system, as well as some peripheral tissues such as pituitary gland and kidney. However, in contrast to mTRP7, which is highly expressed in heart and lung, hTRP7 was undetectable in these tissues. For functional analysis, we heterologously expressed hTRP7 cDNA in an human embryonic kidney cell line. In comparison with untransfected cells depletion of intracellular calcium stores in hTRP7-expressing cells, using either carbachol or thapsigargin, produced a marked increase in the subsequent level of Ca(2+) influx. This increased Ca(2+) entry was blocked by inhibitors of capacitative calcium entry such as La(3+) and Gd(3+). Furthermore, transient transfection of an hTRP7 antisense expression construct into cells expressing hTRP7 eliminated the augmented store-operated Ca(2+) entry. Our findings suggest that hTRP7 is a store-operated calcium channel, a finding in stark contrast to the mouse orthologue, mTRP7, which is reported to enhance Ca(2+) influx independently of store depletion, and suggests that human and mouse TRP7 channels may fulfil different physiological roles.
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- 2002
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25. GABA(B2) is essential for g-protein coupling of the GABA(B) receptor heterodimer.
- Author
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Robbins MJ, Calver AR, Filippov AK, Hirst WD, Russell RB, Wood MD, Nasir S, Couve A, Brown DA, Moss SJ, and Pangalos MN
- Subjects
- Amino Acids genetics, Amino Acids metabolism, Animals, Calcium metabolism, Cells, Cultured, Dimerization, Humans, Kidney cytology, Kidney metabolism, Microinjections, Mutagenesis, Site-Directed, Patch-Clamp Techniques, Protein Binding physiology, Rats, Receptors, GABA genetics, Receptors, GABA metabolism, Receptors, GABA-B genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Analysis, Protein, Signal Transduction physiology, Structure-Activity Relationship, Superior Cervical Ganglion cytology, Superior Cervical Ganglion metabolism, Transfection, GTP-Binding Proteins metabolism, Protein Subunits, Receptors, GABA-B metabolism
- Abstract
GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.
- Published
- 2001
26. Control of progenitor cell number by mitogen supply and demand.
- Author
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van Heyningen P, Calver AR, and Richardson WD
- Subjects
- Animals, Cell Count, Cell Cycle, Cells, Cultured, Dose-Response Relationship, Drug, Embryonic and Fetal Development, Mice, Mitogens pharmacology, Oligodendroglia drug effects, Platelet-Derived Growth Factor pharmacology, Spinal Cord cytology, Stem Cells drug effects, Time Factors, Mitogens metabolism, Oligodendroglia cytology, Platelet-Derived Growth Factor metabolism, Stem Cells cytology
- Abstract
Background: Much is known about how cell proliferation is controlled at the single cell level, but much less about the control of cell numbers in developing populations. Cell number might be determined by an intracellular division limiter or, alternatively, by the availability of mitogens or other factors outside the cell. We investigated the relative importance of intracellular and extracellular controls for one well-defined population of neural precursor cells, namely the glial progenitors that give rise to oligodendrocytes in the mouse spinal cord., Results: We found by cumulative BrdU labeling in vivo that the progenitor cell division cycle slows down markedly as their numbers increase during embryogenesis. When cultured in saturating PDGF, the main mitogen for these cells, their cell cycle accelerated and was independent of their prior rate of division in vivo. This shows that mitogens are limiting in vivo, and suggests that division normally slows down because the PDGF concentration declines. In PDGF-transgenic mice, cell number was proportional to the PDGF supply and apparently unsaturable; at ten times the normal rate of supply, cell number was still increasing but the animals were no longer viable., Conclusions: Progenitor cell proliferation in the embryo is limited by environmental factors, not a cell-intrinsic mechanism. The linear relationship between PDGF supply and final cell number strongly suggests that cells deplete the mitogenic activity in their environment at a rate proportional to the total number of cells. The cells might simply consume the available PDGF or they might secrete autocrine inhibitors, or both.
- Published
- 2001
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27. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.
- Author
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Calver AR, Robbins MJ, Cosio C, Rice SQ, Babbs AJ, Hirst WD, Boyfield I, Wood MD, Russell RB, Price GW, Couve A, Moss SJ, and Pangalos MN
- Subjects
- Amino Acid Motifs physiology, Animals, Cell Line, Cricetinae, Dimerization, GTP-Binding Proteins metabolism, Humans, Mutagenesis, Site-Directed, Protein Structure, Tertiary physiology, Rats, Receptors, Cell Surface metabolism, Receptors, GABA-B genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Transfection, gamma-Aminobutyric Acid metabolism, Intracellular Fluid metabolism, Protein Subunits, Protein Transport physiology, Receptors, GABA-B metabolism, Signal Transduction physiology
- Abstract
GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.
- Published
- 2001
28. Association of GABA(B) receptors and members of the 14-3-3 family of signaling proteins.
- Author
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Couve A, Kittler JT, Uren JM, Calver AR, Pangalos MN, Walsh FS, and Moss SJ
- Subjects
- 14-3-3 Proteins, Animals, Brain Chemistry physiology, COS Cells, Cell Fractionation, Gene Expression physiology, Hippocampus cytology, In Vitro Techniques, Neurons cytology, Neurons metabolism, Protein Structure, Tertiary, Rats, Receptors, GABA-B chemistry, Synapses metabolism, Transfection, Two-Hybrid System Techniques, Receptors, GABA-B genetics, Receptors, GABA-B metabolism, Signal Transduction physiology, Tyrosine 3-Monooxygenase genetics, Tyrosine 3-Monooxygenase metabolism
- Abstract
Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently. Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression. We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1. We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta. We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1. Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3. The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer., (Copyright 2001 Academic Press.)
- Published
- 2001
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29. Comparative immunohistochemical localisation of GABA(B1a), GABA(B1b) and GABA(B2) subunits in rat brain, spinal cord and dorsal root ganglion.
- Author
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Charles KJ, Evans ML, Robbins MJ, Calver AR, Leslie RA, and Pangalos MN
- Subjects
- Animals, Antibody Specificity immunology, Brain cytology, Brain Stem cytology, Brain Stem metabolism, Cerebellum cytology, Cerebellum metabolism, Diencephalon cytology, Diencephalon metabolism, Ganglia, Spinal cytology, Immune Sera immunology, Immunohistochemistry, Male, Neurons cytology, Neurons metabolism, Rats, Rats, Sprague-Dawley, Spinal Cord cytology, Telencephalon cytology, Telencephalon metabolism, Brain metabolism, Ganglia, Spinal metabolism, Receptors, GABA metabolism, Receptors, GABA-B metabolism, Spinal Cord metabolism, gamma-Aminobutyric Acid metabolism
- Abstract
GABA(B) receptors are G-protein-coupled receptors mediating the slow onset and prolonged synaptic actions of GABA in the CNS. The recent cloning of two genes, GABA(B1) and GABA(B2), has revealed a novel requirement for GABA(B) receptor signalling. Studies have demonstrated that the two receptor subunits associate as a GABA(B1)/GABA(B2) heterodimer to form a functional GABA(B) receptor. In this study we have developed polyclonal antisera specific to two splice variants of the GABA(B1) subunit, GABA(B1a) and GABA(B1b), as well as an antiserum to the GABA(B2) subunit. Using affinity-purified antibodies derived from these antisera we have mapped out the distribution profile of each subunit in rat brain, spinal cord and dorsal root ganglion. In brain the highest areas of GABA(B1a), GABA(B1b) and GABA(B2) subunit expression were found in neocortex, hippocampus, thalamus, cerebellum and habenula. In spinal cord, GABA(B1) and GABA(B2) subunits were expressed in the superficial layers of the dorsal horn, as well as in motor neurones in the deeper layers of the ventral horn. GABA(B) receptor subunit immunoreactivity in dorsal root ganglion suggested that expression of GABA(B1b) was restricted to the large diameter neurones, in contrast to GABA(B1a) and GABA(B2) subunits which were expressed in both large and small diameter neurones. Although expression levels of GABA(B1) and GABA(B2) subunits varied we found no areas in which GABA(B1) was expressed in the absence of GABA(B2). This suggests that most, if not all, GABA(B1) immunoreactivity may represent functional GABA(B) receptors. Although our data are in general agreement with functional studies, some discrepancies in GABA(B1) subunit expression occurred with respect to other immunohistochemical studies. Overall our data suggest that GABA(B) receptors are widely expressed throughout the brain and spinal cord, and that GABA(B1a) and GABA(B1b) subunits can associate with GABA(B2) to form both pre- and post-synaptic receptors.
- Published
- 2001
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30. Platelet-derived growth factor is constitutively secreted from neuronal cell bodies but not from axons.
- Author
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Fruttiger M, Calver AR, and Richardson WD
- Subjects
- Animals, Astrocytes cytology, Astrocytes pathology, Astrocytes physiology, Cell Division, Humans, Hyperplasia, Mice, Mice, Knockout, Mice, Transgenic, Neuroglia physiology, Oligodendroglia cytology, Oligodendroglia physiology, Optic Nerve pathology, Optic Nerve physiopathology, Phosphopyruvate Hydratase genetics, Platelet-Derived Growth Factor genetics, Promoter Regions, Genetic, Retina cytology, Axons physiology, Neurons physiology, Optic Nerve physiology, Platelet-Derived Growth Factor metabolism
- Abstract
Neurons synthesise and secrete many growth and survival factors but it is not usually clear whether they are released locally at the cell body or further afield from axons or axon terminals. Without this information, we cannot predict the site(s) of action or the biological functions of many neuron-derived factors. For example, can neuronal platelet-derived growth factor (PDGF) be secreted from axons and reach glial cells in nerve-fibre (white-matter) tracts? To address this question, we expressed PDGF-A in retinal ganglion neurons in transgenic mice and tested for release of PDGF from cell bodies in the retina and from axons in the optic nerve. In both the retina and optic nerve, there are glial cells that express PDGF receptor alpha (PDGFR alpha) [1] and divide in response to PDGF [2-5], so we could detect functional PDGF indirectly through the mitogenic response of glia at both locations. Expressing PDGF-A in neurons under the control of the neuron-specific enolase promoter (NSE-PDGF-A) resulted in a striking hyperplasia of retinal astrocytes, demonstrating that PDGF is secreted from the cell bodies of neurons in the retina [4]. In contrast, glial proliferation in the optic nerve was unaffected, indicating that PDGF is not released from axons. When PDGF was expressed directly in the optic nerve under the control of an astrocyte-specific promoter (GFAP-PDGF-A), oligodendrocyte progenitors hyperproliferated, resulting in a hypertrophic optic nerve. We conclude that PDGF is constitutively secreted from neuronal cell bodies in vivo, but not from axons in white-matter tracts.
- Published
- 2000
- Full Text
- View/download PDF
31. Molecular cloning and characterization of two novel retinoic acid-inducible orphan G-protein-coupled receptors (GPRC5B and GPRC5C).
- Author
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Robbins MJ, Michalovich D, Hill J, Calver AR, Medhurst AD, Gloger I, Sims M, Middlemiss DN, and Pangalos MN
- Subjects
- Amino Acid Sequence, Base Sequence, Brain physiology, Chromosomes, Human, Pair 16, Chromosomes, Human, Pair 17, Cloning, Molecular, DNA Primers chemistry, GTP-Binding Proteins genetics, Gene Expression Regulation, Humans, Molecular Sequence Data, Multigene Family, Phylogeny, RNA, Messenger analysis, Receptors, Cell Surface metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Signal Transduction, Tissue Distribution, Transfection, Tretinoin pharmacology, Tumor Cells, Cultured cytology, Tumor Cells, Cultured drug effects, GTP-Binding Proteins metabolism, Receptors, Cell Surface genetics, Receptors, G-Protein-Coupled
- Abstract
Using homology searching of public databases with a metabotropic glutamate receptor sequence from Caenorhabditis elegans, two novel protein sequences (named RAIG-2 (HGMW-approved symbol GPRC5B) and RAIG-3 (HGMW-approved symbol GPRC5C) were identified containing seven putative transmembrane domains characteristic of G-protein-coupled receptors (GPCRs). RAIG-2 and RAIG-3 encode open reading frames of 403 and 442 amino acid polypeptides, respectively, and show 58% similarity to the recently identified retinoic acid-inducible gene-1 (RAIG-1, HGMW-approved symbol RAI3). Analysis of the three protein sequences places them within the type 3 GPCR family, which includes metabotropic glutamate receptors, GABA(B) receptors, calcium-sensing receptors, and pheromone receptors. However, in contrast to other type 3 GPCRs, RAIG-1, RAIG-2, and RAIG-3 have only short N-terminal domains. RAIG-2 and RAIG-3 cDNA sequences were cloned into the mammalian expression vector pcDNA3 with c-myc or HA epitope tags inserted at their N-termini, respectively. Transient transfection experiments in HEK239T cells using these constructs demonstrated RAIG-2 and RAIG-3 expression at the cell surface. Distribution profiles of mRNA expression obtained by semiquantitative Taq-Man PCR analysis showed RAIG-2 to be predominantly expressed in human brain areas and RAIG-3 to be predominantly expressed in peripheral tissues. In addition, expression of RAIG-2 and RAIG-3 mRNA was increased following treatment with all-trans-retinoic acid in a manner similar to that previously described for RAIG-1. Finally, RAIG-2 was mapped to chromosome 16p12 (D16S405-D16S3045) and RAIG-3 to chromosome 17q25 (D17S1352-D17S785). These results suggest that RAIG-1, RAIG-2, and RAIG-3 represent a novel family of retinoic acid-inducible receptors, most closely related to the type 3 GPCR subfamily, and provide further evidence for a linkage between retinoic acid and G-protein-coupled receptor signal transduction pathways.
- Published
- 2000
- Full Text
- View/download PDF
32. The expression of GABA(B1) and GABA(B2) receptor subunits in the cNS differs from that in peripheral tissues.
- Author
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Calver AR, Medhurst AD, Robbins MJ, Charles KJ, Evans ML, Harrison DC, Stammers M, Hughes SA, Hervieu G, Couve A, Moss SJ, Middlemiss DN, and Pangalos MN
- Subjects
- Animals, Brain metabolism, Female, Humans, Male, Protein Isoforms genetics, Protein Isoforms metabolism, RNA, Messenger metabolism, Rats, Rats, Sprague-Dawley, Receptors, GABA-B genetics, Spinal Cord metabolism, Spleen metabolism, Tissue Distribution, Uterus metabolism, Central Nervous System metabolism, Receptors, GABA-B metabolism
- Abstract
GABA(B) receptors are G-protein-coupled receptors that mediate the slow and prolonged synaptic actions of GABA in the CNS via the modulation of ion channels. Unusually, GABA(B) receptors form functional heterodimers composed of GABA(B1) and GABA(B2) subunits. The GABA(B1) subunit is essential for ligand binding, whereas the GABA(B2) subunit is essential for functional expression of the receptor dimer at the cell surface. We have used real-time reverse transcriptase-polymerase chain reaction to analyse expression levels of these subunits, and their associated splice variants, in the CNS and peripheral tissues of human and rat. GABA(B1) subunit splice variants were expressed throughout the CNS and peripheral tissues, whereas surprisingly GABA(B2) subunit splice variants were neural specific. Using novel antisera specific to individual GABA(B) receptor subunits, we have confirmed these findings at the protein level. Analysis by immunoblotting demonstrated the presence of the GABA(B1) subunit, but not the GABA(B2) subunit, in uterus and spleen. Furthermore, we have shown the first immunocytochemical analysis of the GABA(B2) subunit in the brain and spinal cord using a GABA(B2)-specific antibody. We have, therefore, identified areas of non-overlap between GABA(B1) and GABA(B2) subunit expression in tissues known to contain functional GABA(B) receptors. Such areas are of interest as they may well contain novel GABA(B) receptor subunit isoforms, expression of which would enable the GABA(B1) subunit to reach the cell surface and form functional GABA(B) receptors.
- Published
- 2000
- Full Text
- View/download PDF
33. Defective oligodendrocyte development and severe hypomyelination in PDGF-A knockout mice.
- Author
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Fruttiger M, Karlsson L, Hall AC, Abramsson A, Calver AR, Boström H, Willetts K, Bertold CH, Heath JK, Betsholtz C, and Richardson WD
- Subjects
- Animals, Brain embryology, Cell Differentiation, Cell Division, Mice, Mice, Knockout, Myelin Proteolipid Protein physiology, Platelet-Derived Growth Factor genetics, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins physiology, Proto-Oncogene Proteins c-sis, Receptor, Platelet-Derived Growth Factor alpha, Receptors, Platelet-Derived Growth Factor genetics, Receptors, Platelet-Derived Growth Factor physiology, Central Nervous System embryology, Myelin Sheath physiology, Oligodendroglia physiology, Platelet-Derived Growth Factor physiology
- Abstract
There is a class of oligodendrocyte progenitors, called O-2A progenitors, that is characterized by expression of platelet-derived growth factor &agr;-receptors (PDGFR(&agr;)). It is not known whether all oligodendrocytes are derived from these PDGFRalpha-progenitors or whether a subset(s) of oligodendrocytes develops from a different, PDGFR alpha-negative lineage(s). We investigated the relationship between PDGF and oligodendrogenesis by examining mice that lack either PDGF-A or PDGF-B. PDGF-A null mice had many fewer PDGFR alpha-progenitors than either wild-type or PDGF-B null mice, demonstrating that proliferation of these cells relies heavily (though not exclusively) on PDGF-AA homodimers. PDGF-A-deficient mice also had reduced numbers of oligodendrocytes and a dysmyelinating phenotype (tremor). Not all parts of the central nervous system (CNS) were equally affected in the knockout. For example, there were profound reductions in the numbers of PDGFR alpha-progenitors and oligodendrocytes in the spinal cord and cerebellum, but less severe reductions of both cell types in the medulla. This correlation suggests a close link between PDGFRalpha-progenitors and oligodendrogenesis in most or all parts of the CNS. We also provide evidence that myelin proteolipid protein (PLP/DM-20)-positive cells in the late embryonic brainstem are non-dividing cells, presumably immature oligodendrocytes, and not proliferating precursors.
- Published
- 1999
- Full Text
- View/download PDF
34. Oligodendrocyte population dynamics and the role of PDGF in vivo.
- Author
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Calver AR, Hall AC, Yu WP, Walsh FS, Heath JK, Betsholtz C, and Richardson WD
- Subjects
- Animals, Apoptosis physiology, Cell Differentiation physiology, Cell Division physiology, Dimerization, Female, Gene Expression Regulation, Developmental physiology, Mice, Mice, Transgenic, Myelin Sheath physiology, Platelet-Derived Growth Factor metabolism, Pregnancy, RNA, Messenger analysis, Spinal Cord chemistry, Spinal Cord cytology, Spinal Cord embryology, Stem Cells cytology, Stem Cells physiology, Oligodendroglia cytology, Oligodendroglia physiology, Platelet-Derived Growth Factor genetics
- Abstract
Oligodendrocyte progenitors originate near the floor plate of the spinal cord, then proliferate and migrate throughout the cord before giving rise to oligodendrocytes. Progenitor cell proliferation stops before birth because the cell cycle slows down, linked to an increase in differentiation and death. Experiments with transgenic mice show that platelet-derived growth factor (PDGF) drives progenitor cell division and suggest that slowing of and exit from the cycle reflects a decline in PDGF signaling. Overexpressing PDGF induces hyperproliferation of progenitor cells and excessive, ectopic production of oligodendrocytes. However, the superfluous oligodendrocytes die at an immature stage of differentiation, leaving a normal complement of myelin-forming cells. Therefore, cell survival controls override proliferation controls for determining the final number and distribution of mature oligodendrocytes.
- Published
- 1998
- Full Text
- View/download PDF
35. PDGF mediates a neuron-astrocyte interaction in the developing retina.
- Author
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Fruttiger M, Calver AR, Krüger WH, Mudhar HS, Michalovich D, Takakura N, Nishikawa S, and Richardson WD
- Subjects
- Animals, Animals, Newborn growth & development, Astrocytes cytology, COS Cells, Cell Division, Mice, Mice, Transgenic, Nerve Net drug effects, Neurons cytology, Phenotype, Platelet-Derived Growth Factor metabolism, Rats, Receptor, Platelet-Derived Growth Factor alpha, Receptors, Platelet-Derived Growth Factor antagonists & inhibitors, Receptors, Platelet-Derived Growth Factor metabolism, Retinal Vessels physiology, Astrocytes physiology, Cell Communication physiology, Neurons physiology, Platelet-Derived Growth Factor physiology, Retina growth & development
- Abstract
Astrocytes invade the developing retina from the optic nerve head, over the axons of retinal ganglion cells (RGCs). RGCs express the platelet-derived growth factor A-chain (PDGF-A) and retinal astrocytes the PDGF alpha-receptor (PDGFR alpha), suggesting that PDGF mediates a paracrine interaction between these cells. To test this, we inhibited PDGF signaling in the eye with a neutralizing anti-PDGFR alpha antibody or a soluble extracellular fragment of PDGFR alpha. These treatments inhibited development of the astrocyte network. We also generated transgenic mice that overexpress PDGF-A in RGCs. This resulted in hyperproliferation of astrocytes, which in turn induced excessive vasculogenesis. Thus, PDGF appears to be a link in the chain of cell-cell interactions responsible for matching numbers of neurons, astrocytes, and blood vessels during retinal development.
- Published
- 1996
- Full Text
- View/download PDF
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