G2Cdb::Gene report

Gene id
G00001313
Gene symbol
GRM5 (HGNC)
Species
Homo sapiens
Description
glutamate receptor, metabotropic 5
Orthologue
G00000064 (Mus musculus)

Databases (7)

Gene
ENSG00000168959 (Ensembl human gene)
2915 (Entrez Gene)
96 (G2Cdb plasticity & disease)
GRM5 (GeneCards)
Literature
604102 (OMIM)
Marker Symbol
HGNC:4597 (HGNC)
Protein Sequence
P41594 (UniProt)

Synonyms (3)

  • GPRC1E
  • MGLUR5
  • mGlu5

Diseases (3)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000166: Schizophrenia Y Y (11326300) Microinsertion (MI) Y
D00000166: Schizophrenia Y Y (11326300) Repeat polymorphism (RP) Y
D00000196: Leukodystrophy N Y (14722582) No mutation found (N) N
D00000192: Dystonia parkinsonism (rapid onset) Y Y (15254951) No mutation found (N) N

References

  • Refined linkage to the RDP/DYT12 locus on 19q13.2 and evaluation of GRIK5 as a candidate gene.

    Kamm C, Leung J, Joseph S, Dobyns WB, Brashear A, Breakefield XO and Ozelius LJ

    Molecular Neurogenetics Unit, Department of Neurology, Neuroscience Program, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    By examining two previously described families with rapid-onset dystonia parkinsonism, we have identified a key recombination event that places the disease locus (DYT12) into a 5.9 cM interval flanked by markers D19S224 and D19S900. Evaluation of a positional candidate gene, the glutamate receptor subunit GRIK5, revealed no mutations.

    Funded by: NINDS NIH HHS: NS38372

    Movement disorders : official journal of the Movement Disorder Society 2004;19;7;845-7

  • Molecular characterization of an 11q14.3 microdeletion associated with leukodystrophy.

    Goizet C, Coupry I, Rooryck C, Taine L, Dormoy V, Lacombe D and Arveiler B

    Laboratoire de Génétique Humaine, Développement et Cancer, Université Victor Segalen Bordeaux 2, F-33076, Bordeaux, France. cyril.goizet@chu-bordeaux.fr

    Leukodystrophies represent a heterogeneous group of rare hereditary diseases affecting the central nervous system. The underlying molecular defect remains unknown in almost 50% of cases. We previously assigned a new locus for leukodystrophy of unknown cause to chromosome 11q14.3 by identifying a de novo microdeletion in a sporadic case. We now report the precise molecular characterization of this microdeletion. Physical mapping of the region of interest allowed us to identify and analyze candidate gene(s) possibly implicated in leukodystrophy.

    European journal of human genetics : EJHG 2004;12;3;245-50

  • The genomic organisation of the metabotropic glutamate receptor subtype 5 gene, and its association with schizophrenia.

    Devon RS, Anderson S, Teague PW, Muir WJ, Murray V, Pelosi AJ, Blackwood DH and Porteous DJ

    Medical Genetics Section, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK.

    The G-protein coupled metabotropic glutamate receptors (GRMs/mGluRs) have been implicated in the aetiology of schizophrenia as they modulate the NMDA response and that of other neurotransmitters including dopamine and GABA.(1-3) Electrophysiological studies in GRM subtype 5 knockout mice reveal, in one study, a sensorimotor gating deficit characteristic of schizophrenia and in another, a key rôle for this gene in the modulation of hippocampal NMDA-dependent synaptic plasticity. In humans, GRM5 levels are increased in certain pyramidal cell neurons in schizophrenics vs controls.(6) Finally, GRM5 has been mapped to 11q14, neighbouring a translocation that segregates with schizophrenia and related psychoses in a large Scottish family, F23 (MLOD score 6.0). We determined the intron/exon structure of GRM5 and identified a novel intragenic microsatellite. A case-control association study identified a significant difference in allele frequency distribution between schizophrenics and controls (P = 0.04). This is suggestive of involvement of the GRM5 gene in schizophrenia in this population.

    Molecular psychiatry 2001;6;3;311-4

Literature (42)

Pubmed - human_disease

  • Molecular characterization of an 11q14.3 microdeletion associated with leukodystrophy.

    Goizet C, Coupry I, Rooryck C, Taine L, Dormoy V, Lacombe D and Arveiler B

    Laboratoire de Génétique Humaine, Développement et Cancer, Université Victor Segalen Bordeaux 2, F-33076, Bordeaux, France. cyril.goizet@chu-bordeaux.fr

    Leukodystrophies represent a heterogeneous group of rare hereditary diseases affecting the central nervous system. The underlying molecular defect remains unknown in almost 50% of cases. We previously assigned a new locus for leukodystrophy of unknown cause to chromosome 11q14.3 by identifying a de novo microdeletion in a sporadic case. We now report the precise molecular characterization of this microdeletion. Physical mapping of the region of interest allowed us to identify and analyze candidate gene(s) possibly implicated in leukodystrophy.

    European journal of human genetics : EJHG 2004;12;3;245-50

  • The genomic organisation of the metabotropic glutamate receptor subtype 5 gene, and its association with schizophrenia.

    Devon RS, Anderson S, Teague PW, Muir WJ, Murray V, Pelosi AJ, Blackwood DH and Porteous DJ

    Medical Genetics Section, University of Edinburgh, Molecular Medicine Centre, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK.

    The G-protein coupled metabotropic glutamate receptors (GRMs/mGluRs) have been implicated in the aetiology of schizophrenia as they modulate the NMDA response and that of other neurotransmitters including dopamine and GABA.(1-3) Electrophysiological studies in GRM subtype 5 knockout mice reveal, in one study, a sensorimotor gating deficit characteristic of schizophrenia and in another, a key rôle for this gene in the modulation of hippocampal NMDA-dependent synaptic plasticity. In humans, GRM5 levels are increased in certain pyramidal cell neurons in schizophrenics vs controls.(6) Finally, GRM5 has been mapped to 11q14, neighbouring a translocation that segregates with schizophrenia and related psychoses in a large Scottish family, F23 (MLOD score 6.0). We determined the intron/exon structure of GRM5 and identified a novel intragenic microsatellite. A case-control association study identified a significant difference in allele frequency distribution between schizophrenics and controls (P = 0.04). This is suggestive of involvement of the GRM5 gene in schizophrenia in this population.

    Molecular psychiatry 2001;6;3;311-4

Pubmed - other

  • Common genetic variation and the control of HIV-1 in humans.

    Fellay J, Ge D, Shianna KV, Colombo S, Ledergerber B, Cirulli ET, Urban TJ, Zhang K, Gumbs CE, Smith JP, Castagna A, Cozzi-Lepri A, De Luca A, Easterbrook P, Günthard HF, Mallal S, Mussini C, Dalmau J, Martinez-Picado J, Miro JM, Obel N, Wolinsky SM, Martinson JJ, Detels R, Margolick JB, Jacobson LP, Descombes P, Antonarakis SE, Beckmann JS, O'Brien SJ, Letvin NL, McMichael AJ, Haynes BF, Carrington M, Feng S, Telenti A, Goldstein DB and NIAID Center for HIV/AIDS Vaccine Immunology (CHAVI)

    Center for Human Genome Variation, Duke Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, USA.

    To extend the understanding of host genetic determinants of HIV-1 control, we performed a genome-wide association study in a cohort of 2,554 infected Caucasian subjects. The study was powered to detect common genetic variants explaining down to 1.3% of the variability in viral load at set point. We provide overwhelming confirmation of three associations previously reported in a genome-wide study and show further independent effects of both common and rare variants in the Major Histocompatibility Complex region (MHC). We also examined the polymorphisms reported in previous candidate gene studies and fail to support a role for any variant outside of the MHC or the chemokine receptor cluster on chromosome 3. In addition, we evaluated functional variants, copy-number polymorphisms, epistatic interactions, and biological pathways. This study thus represents a comprehensive assessment of common human genetic variation in HIV-1 control in Caucasians.

    Funded by: CCR NIH HHS: HHSN261200800001C; Intramural NIH HHS; Medical Research Council: G0200585; NCI NIH HHS: HHSN261200800001E; NIAID NIH HHS: AI067854, U01 AI067854, U19 AI067854; PHS HHS: HHSN261200800001E

    PLoS genetics 2009;5;12;e1000791

  • The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

    Canela L, Fernández-Dueñas V, Albergaria C, Watanabe M, Lluís C, Mallol J, Canela EI, Franco R, Luján R and Ciruela F

    IDIBAPS, CIBERNED and Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.

    Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

    Journal of neurochemistry 2009;111;2;555-67

  • Identification of new putative susceptibility genes for several psychiatric disorders by association analysis of regulatory and non-synonymous SNPs of 306 genes involved in neurotransmission and neurodevelopment.

    Gratacòs M, Costas J, de Cid R, Bayés M, González JR, Baca-García E, de Diego Y, Fernández-Aranda F, Fernández-Piqueras J, Guitart M, Martín-Santos R, Martorell L, Menchón JM, Roca M, Sáiz-Ruiz J, Sanjuán J, Torrens M, Urretavizcaya M, Valero J, Vilella E, Estivill X, Carracedo A and Psychiatric Genetics Network Group

    CIBER en Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain.

    A fundamental difficulty in human genetics research is the identification of the spectrum of genetic variants that contribute to the susceptibility to common/complex disorders. We tested here the hypothesis that functional genetic variants may confer susceptibility to several related common disorders. We analyzed five main psychiatric diagnostic categories (substance-abuse, anxiety, eating, psychotic, and mood disorders) and two different control groups, representing a total of 3,214 samples, for 748 promoter and non-synonymous single nucleotide polymorphisms (SNPs) at 306 genes involved in neurotransmission and/or neurodevelopment. We identified strong associations to individual disorders, such as growth hormone releasing hormone (GHRH) with anxiety disorders, prolactin regulatory element (PREB) with eating disorders, ionotropic kainate glutamate receptor 5 (GRIK5) with bipolar disorder and several SNPs associated to several disorders, that may represent individual and related disease susceptibility factors. Remarkably, a functional SNP, rs945032, located in the promoter region of the bradykinin receptor B2 gene (BDKRB2) was associated to three disorders (panic disorder, substance abuse, and bipolar disorder), and two additional BDKRB2 SNPs to obsessive-compulsive disorder and major depression, providing evidence for common variants of susceptibility to several related psychiatric disorders. The association of BDKRB2 (odd ratios between 1.65 and 3.06) to several psychiatric disorders supports the view that a common genetic variant could confer susceptibility to clinically related phenotypes, and defines a new functional hint in the pathophysiology of psychiatric diseases.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;6;808-16

  • Genome-wide association study of tanning phenotype in a population of European ancestry.

    Nan H, Kraft P, Qureshi AA, Guo Q, Chen C, Hankinson SE, Hu FB, Thomas G, Hoover RN, Chanock S, Hunter DJ and Han J

    Channing Laboratory, Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA. hnan@hsph.harvard.edu

    We conducted a multistage genome-wide association study (GWAS) of tanning response after exposure to sunlight in over 9,000 men and women of European ancestry who live in the United States. An initial analysis of 528,173 single-nucleotide polymorphisms (SNPs) genotyped on 2,287 women identified LOC401937 (rs966321) on chromosome 1 as a novel locus highly associated with tanning ability, and we confirmed this association in 870 women controls from a skin cancer case-control study with joint P-value=1.6 x 10(-9). We further genotyped this SNP in two subsequent replication studies (one with 3,750 women and the other with 2,405 men). This association was not replicated in either of these two studies. We found that several SNPs reaching the genome-wide significance level are located in or adjacent to the loci previously known as pigmentation genes: MATP, IRF4, TYR, OCA2, and MC1R. Overall, these tanning ability-related loci are similar to the hair color-related loci previously reported in the GWAS of hair color.

    Funded by: NCI NIH HHS: CA122838, CA128080, R01 CA122838, R01 CA122838-01A2, R03 CA128080, R03 CA128080-02

    The Journal of investigative dermatology 2009;129;9;2250-7

  • Pharmacogenetics of antipsychotic response in the CATIE trial: a candidate gene analysis.

    Need AC, Keefe RS, Ge D, Grossman I, Dickson S, McEvoy JP and Goldstein DB

    Center for Human Genome Variation, Institute for Genome Sciences & Policy, Duke University, Durham, NC 27708, USA.

    The Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) Phase 1 Schizophrenia trial compared the effectiveness of one typical and four atypical antipsychotic medications. Although trials such as CATIE present important opportunities for pharmacogenetics research, the very richness of the clinical data presents challenges for statistical interpretation, and in particular the risk that data mining will lead to false-positive discoveries. For this reason, it is both misleading and unhelpful to perpetuate the current practice of reporting association results for these trials one gene at a time, ignoring the fact that multiple gene-by-phenotype tests are being carried out on the same data set. On the other hand, suggestive associations in such trials may lead to new hypotheses that can be tested through both replication efforts and biological experimentation. The appropriate handling of these forms of data therefore requires dissemination of association statistics without undue emphasis on select findings. Here we attempt to illustrate this approach by presenting association statistics for 2769 polymorphisms in 118 candidate genes evaluated for 21 pharmacogenetic phenotypes. On current evidence it is impossible to know which of these associations may be real, although in total they form a valuable resource that is immediately available to the scientific community.

    Funded by: NIMH NIH HHS: N01 MH90001

    European journal of human genetics : EJHG 2009;17;7;946-57

  • Antagonists at metabotropic glutamate receptor subtype 5: structure activity relationships and therapeutic potential for addiction.

    Carroll FI

    Center for Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, NC 27709-2194, USA. fic@rti.org

    As a result of intensive investigation, particularly in the pharmaceutical industry, a number of potent and selective metabotropic glutamate receptor subtype 5 (mGluR5) antagonists have been discovered. The structure activity relationship studies that led to the discovery of these mGluR5 antagonists are presented in this review. Results from studies on selected mGluR5 antagonists in animal models that simulate drug reward, reinforcement, and relapse appear promising. The comorbidity between drug abuse and anxiety and depression make drugs active in these disorders of great interest. Clinical studies showed that the mGluR5 antagonist fenobam was an active anxiolytic drug. Several new mGluR5 antagonists produced anxiolytic and antidepressant-like effects in animal models of these disorders. The results from the clinical and animal studies provide information for new approaches to finding mechanistically distinct pharmacotherapies to help patients achieve and maintain abstinence from cocaine, methamphetamine, opiates, ethanol, and nicotine (smoking).

    Annals of the New York Academy of Sciences 2008;1141;221-32

  • Essential role for epidermal growth factor receptor in glutamate receptor signaling to NF-kappaB.

    Sitcheran R, Comb WC, Cogswell PC and Baldwin AS

    Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA. sitcheran@unc.edu

    Glutamate is a critical neurotransmitter of the central nervous system (CNS) and also an important regulator of cell survival and proliferation. The binding of glutamate to metabotropic glutamate receptors induces signal transduction cascades that lead to gene-specific transcription. The transcription factor NF-kappaB, which regulates cell proliferation and survival, is activated by glutamate; however, the glutamate receptor-induced signaling pathways that lead to this activation are not clearly defined. Here we investigate the glutamate-induced activation of NF-kappaB in glial cells of the CNS, including primary astrocytes. We show that glutamate induces phosphorylation, nuclear accumulation, DNA binding, and transcriptional activation function of glial p65. The glutamate-induced activation of NF-kappaB requires calcium-dependent IkappaB kinase alpha (IKKalpha) and IKKbeta activation and induces p65-IkappaBalpha dissociation in the absence of IkappaBalpha phosphorylation or degradation. Moreover, glutamate-induced IKK preferentially targets the phosphorylation of p65 but not IkappaBalpha. Finally, we show that the ability of glutamate to activate NF-kappaB requires cross-coupled signaling with the epidermal growth factor receptor. Our results provide insight into a glutamate-induced regulatory pathway distinct from that described for cytokine-induced NF-kappaB activation and have important implications with regard to both normal glial cell physiology and pathogenesis.

    Funded by: NCI NIH HHS: CA73756, CA75080, K01 CA118274, K01 CA118274-03, KO1-CA118274, R01 CA073756, R01 CA075080; NIAID NIH HHS: AI35098, R01 AI035098, R37 AI035098

    Molecular and cellular biology 2008;28;16;5061-70

  • Systematic analysis of glutamatergic neurotransmission genes in alcohol dependence and adolescent risky drinking behavior.

    Schumann G, Johann M, Frank J, Preuss U, Dahmen N, Laucht M, Rietschel M, Rujescu D, Lourdusamy A, Clarke TK, Krause K, Dyer A, Depner M, Wellek S, Treutlein J, Szegedi A, Giegling I, Cichon S, Blomeyer D, Heinz A, Heath S, Lathrop M, Wodarz N, Soyka M, Spanagel R and Mann K

    Interdisciplinary Research Group Addiction, MRC-SGDP Center, Institute of Psychiatry at King's College, POB 080, London SE5 8AF, England. g.schumann@iop.kcl.ac.uk

    Context: Glutamatergic neurotransmission is implicated in alcohol-drinking behavior in animal models.

    Objective: To investigate whether genetic variations in glutamatergic neurotransmission genes, which are known to alter alcohol effects in rodents, contribute to the genetic basis of alcoholism in humans.

    Design: Association analysis of alcohol dependence and haplotype-tagging single nucleotide polymorphisms (SNPs) covering 10 glutamatergic genes. Resequencing of functional domains of these genes identified 204 SNPs. Haplotypes with a frequency of 5% or greater could be discriminated by 21 haplotype-tagging SNPs analyzed for association in 2 independent samples of alcohol-dependent adult patients and controls as well as adolescent trios.

    Setting: Four university medical centers in the south of Germany.

    Participants: One thousand three hundred thirty-seven patients and 1555 controls (study 1: 544 patients, 553 controls; study 2: 793 patients, 1002 controls). One hundred forty-four trios of 15-year-old adolescents assessed for risky drinking behavior.

    Genotype profiles for GLAST; N-methyl-d-aspartate-receptor subunits NR1, NR2A, and NR2B; MGLUR5; NNOS; PRKG2; CAMK4; the regulatory subunit of PI3K; and CREB were analyzed for association with alcohol dependence using multivariate statistical analysis. Risky adolescent drinking was tested using the transmission disequilibrium test.

    Results: Analysis of study 1 revealed that NR2A and MGLUR5 have the greatest relevance for human alcohol dependence among the genes selected with odds ratios of 2.35 and 1.69, respectively. Replication analysis in study 2 confirmed an association of alcohol dependence with NR2A (odds ratio, 2.01) but showed no association with MGLUR5. Combined analysis of study 1 and study 2 exhibited a more significant association on the Cochran-Mantel-Haenszel test (P < .001) for NR2A; NR2A was associated with positive family history, early onset of alcoholism, and maximum number of drinks in adults as well as risky drinking patterns in adolescents.

    Conclusion: Genetic variations in NR2A have the greatest relevance for human alcohol dependence among the glutamatergic genes selected for their known alteration of alcohol effects in animal models.

    Funded by: Department of Health

    Archives of general psychiatry 2008;65;7;826-38

  • Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear Gq/11 proteins to generate inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ release.

    Kumar V, Jong YJ and O'Malley KL

    Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    Recently we have shown that the metabotropic glutamate 5 (mGlu5) receptor can be expressed on nuclear membranes of heterologous cells or endogenously on striatal neurons where it can mediate nuclear Ca2+ changes. Here, pharmacological, optical, and genetic techniques were used to show that upon activation, nuclear mGlu5 receptors generate nuclear inositol 1,4,5-trisphosphate (IP3) in situ. Specifically, expression of an mGlu5 F767S mutant in HEK293 cells that blocks Gq/11 coupling or introduction of a dominant negative Galphaq construct in striatal neurons prevented nuclear Ca2+ changes following receptor activation. These data indicate that nuclear mGlu5 receptors couple to Gq/11 to mobilize nuclear Ca2+. Nuclear mGlu5-mediated Ca2+ responses could also be blocked by the phospholipase C (PLC) inhibitor, U73122, the phosphatidylinositol (PI) PLC inhibitor 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3), or by using small interfering RNA targeted against PLCbeta1 demonstrating that PI-PLC is involved. Direct assessment of inositol phosphate production using a PIP2/IP3 "biosensor" revealed for the first time that IP3 can be generated in the nucleus following activation of nuclear mGlu5 receptors. Finally, both IP3 and ryanodine receptor blockers prevented nuclear mGlu5-mediated increases in intranuclear Ca2+. Collectively, this study shows that like plasma membrane receptors, activated nuclear mGlu5 receptors couple to Gq/11 and PLC to generate IP3-mediated release of Ca2+ from Ca2+-release channels in the nucleus. Thus the nucleus can function as an autonomous organelle independent of signals originating in the cytoplasm, and nuclear mGlu5 receptors play a dynamic role in mobilizing Ca2+ in a specific, localized fashion.

    Funded by: NIMH NIH HHS: MH57817, MH69646; NINDS NIH HHS: NS057105

    The Journal of biological chemistry 2008;283;20;14072-83

  • Direct interaction enables cross-talk between ionotropic and group I metabotropic glutamate receptors.

    Perroy J, Raynaud F, Homburger V, Rousset MC, Telley L, Bockaert J and Fagni L

    Institut de Génomique Fonctionnelle, CNRS UMR 5203, INSERM U661, Universités de Montpellier 1 and 2, Montpellier 34094, France.

    Functional interplay between ionotropic and metabotropic receptors frequently involves complex intracellular signaling cascades. The group I metabotropic glutamate receptor mGlu5a co-clusters with the ionotropic N-methyl-d-aspartate (NMDA) receptor in hippocampal neurons. In this study, we report that a more direct cross-talk can exist between these types of receptors. Using bioluminescence resonance energy transfer in living HEK293 cells, we demonstrate that mGlu5a and NMDA receptor clustering reflects the existence of direct physical interactions. Consequently, the mGlu5a receptor decreased NMDA receptor current, and reciprocally, the NMDA receptor strongly reduced the ability of the mGlu5a receptor to release intracellular calcium. We show that deletion of the C terminus of the mGlu5a receptor abolished both its interaction with the NMDA receptor and reciprocal inhibition of the receptors. This direct functional interaction implies a higher degree of target-effector specificity, timing, and subcellular localization of signaling than could ever be predicted with complex signaling pathways.

    The Journal of biological chemistry 2008;283;11;6799-805

  • Species differences in mGluR5 binding sites in mammalian central nervous system determined using in vitro binding with [18F]F-PEB.

    Patel S, Hamill TG, Connolly B, Jagoda E, Li W and Gibson RE

    Department of Research Imaging, Merck Research Laboratories, West Point, PA 19486, USA. shailendra_patel@merck.com

    Binding of [18F]3-fluoro-5-[(pyridin-3-yl)ethynyl]benzonitrile ([18F]F-PEB) was evaluated in membranes and tissue sections prepared from rat, rhesus and human brain. Saturation equilibrium binding experiments with frozen brain cortex and caudate-putamen membranes of young adult rhesus and human and with cortex and striatum from rat yielded data indicative of specific high-affinity binding (KD=0.1-0.15 nM, n> or =3) to a saturable site previously shown to be metabotropic glutamate receptor 5 (mGluR5; Patel S, Ndubizu O, Hamill T, Chaudhary A, Burns HD, Hargreaves RJ, Gibson RE. Screening cascade and development of potential positron emission tomography radiotracers for mGluR5: in vitro and in vivo characterization. Mol Imaging Biol 2005;7:314-323). High-affinity binding of [18F]F-PEB was also detected in cerebellum membranes from rat, rhesus and human. The density of binding sites (Bmax) measured using [18F]F-PEB followed the rank order cortex approximately caudate-putamen/striatum>cerebellum for all three species, with the cerebellum Bmax being significantly lower than that observed in the other regions. Receptor autoradiography studies in tissue sections confirmed that the regional distribution of [18F]F-PEB in mammalian central nervous system is consistent with that of mGluR5 and that a small but specific mGluR5 signal is observed in rhesus and human cerebellum. A small and quantifiable specific signal could also be observed in rat cerebellum using this radiotracer. Immunohistochemical analysis in brain sections revealed a rank order of staining in rhesus and human brain of cortex approximately caudate-putamen>cerebellum. Rat brain immunohistochemistry followed the same rank order, although the staining in the cerebellum was significantly lower. Using a "no-wash" wipe assay, the development of a specific signal within 20 min of incubation of tissue brain sections (>60% in the cortex and striatum; 36-49% in the cerebellum) from all three species confirmed previous in vivo data from rat and rhesus monkey that [18F]PEB is likely to provide a useful in vivo signal using positron emission tomography (PET). This study provides the first quantitative demonstration and direct comparison of a PET tracer candidate identifying mGluR5 binding sites in mammalian cerebellum, which subsequently raises questions in terms of using the cerebellum as a null tissue in PET imaging studies in the laboratory and the clinic.

    Nuclear medicine and biology 2007;34;8;1009-17

  • Actin-binding protein alpha-actinin-1 interacts with the metabotropic glutamate receptor type 5b and modulates the cell surface expression and function of the receptor.

    Cabello N, Remelli R, Canela L, Soriguera A, Mallol J, Canela EI, Robbins MJ, Lluis C, Franco R, McIlhinney RA and Ciruela F

    Institut d'Investigacions Biomèdiques August Pi i Sunyer and Department of Biochemistry and Molecular Biology, University of Barcelona, Facultat de Biologia, Avda. Diagonal 645, Barcelona 08028, Spain.

    Receptors for neurotransmitters require scaffolding proteins for membrane microdomain targeting and for regulating receptor function. Using a yeast two-hybrid screen, alpha-actinin-1, a major F-actin cross-linking protein, was identified as a binding partner for the C-terminal domain of metabotropic glutamate receptor type 5b (mGlu(5b) receptor). Co-expression, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between mGlu(5b) receptor and alpha-actinin-1 in both transfected HEK-293 cells and rat striatum. The interaction of alpha-actinin-1 with mGlu(5b) receptor modulated the cell surface expression of the receptor. This was dependent on the binding of alpha-actinin-1 to the actin cytoskeleton. In addition, the alpha-actinin-1/mGlu(5b) receptor interaction regulated receptor-mediated activation of the mitogen-activated protein kinase pathway. Together, these findings indicate that there is an alpha-actinin-1-dependent mGlu(5b) receptor association with the actin cytoskeleton modulating receptor cell surface expression and functioning.

    Funded by: Medical Research Council: MC_U138162357

    The Journal of biological chemistry 2007;282;16;12143-53

  • Regulation of the mGluR5, EAAT1 and GS expression by glucocorticoids in MG-63 osteoblast-like osteosarcoma cells.

    Kalariti N, Lembessis P, Papageorgiou E, Pissimissis N and Koutsilieris M

    Department of Experimental Physiology, Medical School, University of Athens, Goudi-Athens, Greece.

    Introduction: Growth factors, cytokines, sex steroid hormones and glucocorticoids have differential and complex effects on skeletal metabolism. Recently, the presence of the glutamatergic (Glu) system in bone cells has provided new evidence for its possible role in bone physiology. Consequently, we have investigated the regulation of certain components of the Glu system by glucocorticoids in MG-63 osteoblast-like osteosarcoma cells, in vitro.

    We characterized the effects of dexamethasone on the expression of the mGluR5, EAAT1 and GS, at mRNA and protein level, using relative quantitative RTPCR and Western blot analysis, respectively.

    Results: We confirmed the induction of GS expression by dexamethasone published previously. In addition, we documented for the first time the expression of the mGluR5 and EAAT1 in MG-63 cells, as well as the ability of dexamethasone to upregulate the expression of the mGluR5 and EAAT1 in the MG-63 cells.

    Conclusions: Components of the glutamatergic system may play a role in bone pathophysiology.

    Journal of musculoskeletal & neuronal interactions 2007;7;2;113-8

  • Protein kinase C phosphorylation of the metabotropic glutamate receptor mGluR5 on Serine 839 regulates Ca2+ oscillations.

    Kim CH, Braud S, Isaac JT and Roche KW

    NINDS, National Institutes of Health, Bethesda, Maryland 20892, USA.

    The activation of Group 1 metabotropic glutamate receptors, mGluR5 and mGluR1alpha, triggers intracellular calcium release; however, mGluR5 activation is unique in that it elicits Ca2+ oscillations. A short region of the mGluR5 C terminus is the critical determinant and differs from the analogous region of mGluR1alpha by a single amino acid residue, Thr-840, which is an aspartic acid (Asp-854) in mGluR1alpha. Previous studies show that mGluR5-elicited Ca2+ oscillations require protein kinase C (PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylation site. However, direct phosphorylation of mGluR5 has not been studied in detail. We have used biochemical analyses to directly investigate the phosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 is directly phosphorylated by PKC, whereas Thr-840 plays a permissive role. Although Ser-839 is conserved in mGluR1alpha (Ser-853), it is not phosphorylated, as the adjacent residue (Asp-854) is not permissive; however, mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylation of Ser-853 on mGluR1alpha. We investigated the physiological consequences of mGluR5 Ser-839 phosphorylation using Ca2+ imaging. Mutations that eliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependent Ca2+ oscillations. However, mutation of Thr-840 to alanine, which prevents potential Thr-840 phosphorylation but is still permissive for Ser-839 phosphorylation, has no effect on Ca2+ oscillations. Thus, we showed that it is phosphorylation of Ser-839, not Thr-840, that is absolutely required for the unique Ca2+ oscillations produced by mGluR5 activation. The Thr-840 residue is important only in that it is permissive for the PKC-dependent phosphorylation of Ser-839.

    Funded by: Wellcome Trust

    The Journal of biological chemistry 2005;280;27;25409-15

  • Interactions between ephrin-B and metabotropic glutamate 1 receptors in brain tissue and cultured neurons.

    Calò L, Bruno V, Spinsanti P, Molinari G, Korkhov V, Esposito Z, Patanè M, Melchiorri D, Freissmuth M and Nicoletti F

    Department of Human Physiology and Pharmacology, University of Rome La Sapienza, 00185 Rome, Italy.

    We examined the interaction between ephrins and metabotropic glutamate (mGlu) receptors in the developing brain and cultured neurons. EphrinB2 coimmunoprecipitated with mGlu1a receptors, in all of the brain regions examined, and with mGlu5 receptors in the corpus striatum. In striatal slices, activation of ephrinB2 by a clustered form of its target receptor, EphB1, amplified the mGlu receptor-mediated stimulation of polyphosphoinositide (PI) hydrolysis. This effect was abolished in slices treated with mGlu1 or NMDA receptor antagonists but was not affected by pharmacological blockade of mGlu5 receptors. An interaction among ephrinB2, mGlu1 receptor, and NMDA was supported by the following observations: (1) the NR1 subunit of NMDA receptors coimmunoprecipitated with mGlu1a receptors and ephrinB2 in striatal lysates; (2) clustered EphB1 amplified excitatory amino acid-stimulated PI hydrolysis in cultured granule cells grown under conditions that favored the expression of mGlu1a receptors; and (3) clustered EphB1 amplified the enhancing effect of mGlu receptor agonists on NMDA toxicity in cortical cultures, and its action was sensitive to mGlu1 receptor antagonists. Finally, fluorescence resonance energy transfer and coclustering analysis in human embryonic kidney 293 cells excluded a physical interaction between ephrinB2 and mGlu1a (or mGlu5 receptors). A functional interaction between ephrinB and mGlu1 receptors, which likely involves adaptor or scaffolding proteins, might have an important role in the regulation of developmental plasticity.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;9;2245-54

  • A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP).

    Heydorn A, Søndergaard BP, Ersbøll B, Holst B, Nielsen FC, Haft CR, Whistler J and Schwartz TW

    Laboratory for Molecular Pharmacology, Department of Pharmacology, Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.

    Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor proteins previously proposed to be involved in post-endocytotic sorting of receptors. Of the two proteins suggested to target receptors for recycling to the cell membrane, which is the route believed to be taken by a majority of receptors, ERM (ezrin-radixin-moesin)-binding phosphoprotein 50 (EBP50) bound only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein-coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the delta-opioid receptor, confirmed the expected nanomolar affinities for interaction with SNX1. Truncations of the NK(1) receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrum-recycling adaptor.

    The Journal of biological chemistry 2004;279;52;54291-303

  • Group I metabotropic glutamate receptors mediate a dual role of glutamate in T cell activation.

    Pacheco R, Ciruela F, Casadó V, Mallol J, Gallart T, Lluis C and Franco R

    Department of Biochemistry and Molecular Biology, University of Barcelona, 08028 Barcelona, Spain.

    Metabotropic glutamate receptors (mGluR) are present in cells of the nervous system, where they are activated by one of the main neurotransmitters, glutamate. They are also expressed in cells outside the nervous system. We identified and characterized two receptors belonging to group I mGluR, mGlu1R and mGlu5R, in human cell lines of lymphoid origin and in resting and activated lymphocytes from human peripheral blood. Both are highly expressed in the human Jurkat T cell line, whereas mGlu5R is expressed only in the human B cell line SKW6.4. In blood lymphocytes, mGlu5R is expressed constitutively, whereas mGlu1R is expressed only upon activation via the T cell receptor-CD3 complex. Group I receptors in the central nervous system are coupled to phospholipase C, whereas in blood lymphocytes, activation of mGlu5R does not trigger this signaling pathway, but instead activates adenylate cyclase. On the other hand, mGlu5R does not mediate ERK1/2 activation, whereas mGlu1R, which is coupled neither to phospholipase C nor to calcium channels and whose activation does not increase cAMP, activates the mitogen-activated protein kinase cascade. The differential expression of mGluR in resting and activated lymphocytes and the different signaling pathways that are triggered when mGlu1Rs or mGlu5Rs are activated point to a key role of glutamate in the regulation of T cell physiological function. The study of the signaling pathways (cAMP production and ERK1/2 phosphorylation) and the proliferative response obtained in the presence of glutamate analogs suggests that mGlu1R and mGlu5R have distinct functions. mGlu5R mediates the reported inhibition of cell proliferation evoked by glutamate, which is reverted by the activation of inducible mGlu1R. This is a novel non-inhibitory action mechanism for glutamate in lymphocyte activation. mGlu1R and mGlu5R thus mediate opposite glutamate effects in human lymphocytes.

    The Journal of biological chemistry 2004;279;32;33352-8

  • Refined linkage to the RDP/DYT12 locus on 19q13.2 and evaluation of GRIK5 as a candidate gene.

    Kamm C, Leung J, Joseph S, Dobyns WB, Brashear A, Breakefield XO and Ozelius LJ

    Molecular Neurogenetics Unit, Department of Neurology, Neuroscience Program, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    By examining two previously described families with rapid-onset dystonia parkinsonism, we have identified a key recombination event that places the disease locus (DYT12) into a 5.9 cM interval flanked by markers D19S224 and D19S900. Evaluation of a positional candidate gene, the glutamate receptor subunit GRIK5, revealed no mutations.

    Funded by: NINDS NIH HHS: NS38372

    Movement disorders : official journal of the Movement Disorder Society 2004;19;7;845-7

  • Metabotropic glutamate receptors are expressed in adult human glial progenitor cells.

    Luyt K, Varadi A, Halfpenny CA, Scolding NJ and Molnar E

    MRC Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.

    Glial precursor cells (GPCs) are present in the adult human central nervous system (CNS) and they can be isolated and maintained in culture for in vitro studies. This study analysed expression of mGluR3 and mGluR5 metabotropic glutamate receptor (mGluR) mRNAs in GPCs. A2B5 surface antigen positive GPCs were isolated using immunomagnetic selection from dissociated temporal lobe subcortical white matter cells. The separated GPCs were maintained in cultures and characterised by immunoreactivity for the differentiation markers A2B5 and human platelet-derived growth factor-alpha receptor (PDGFalphaR). Reverse transcription followed by multiplex PCR analysis showed that the GPCs expressed both mGluR3 and mGluR5a mRNAs. Double immunostaining for glial progenitor markers and mGluR5 proteins demonstrated that all A2B5 and PDGFalphaR-positive cells were also positive for mGluR5. The results indicate that GPCs present in the adult human CNS express mGluR3 and mGluR5a. These neurotransmitter receptors may be involved in the proliferation and differentiation of glial cells.

    Biochemical and biophysical research communications 2004;319;1;120-9

  • Differential expression of mGluR5 in human lumbosacral motoneurons.

    Anneser JM, Ince PG, Shaw PJ and Borasio GD

    Department of Neurology, Ludwig-Maximilians-University, Klinikum Grosshadern, Marchioninistr. 15, 81377 Munich, Germany. janneser@brain.nefo.med.uni-muenche.de

    Glutamatergic excitotoxicity is one of the main hypotheses to explain motoneuronal degeneration in amyotrophic lateral sclerosis (ALS). Interestingly, autonomic motoneurons remain almost unaffected, even in late stages of the disease. Since glutamate receptors may mediate neurotoxic as well as neuroprotective effects, different expression patterns may contribute to neuronal vulnerability. We and others have previously described a significantly higher expression of group I metabotropic glutamate receptors (mGluRs) in rat autonomic motoneurons compared to somatic motoneurons. Here we show a selective expression of the group I receptor mGluR5 in human parasympathetic Onuf's nucleus. These results are in accordance with previous findings in rat and strengthen the hypothesis that mGluR expression may provide a possible clue to the selective vulnerability in ALS.

    Neuroreport 2004;15;2;271-3

  • Isoform-specific phosphorylation of metabotropic glutamate receptor 5 by protein kinase C (PKC) blocks Ca2+ oscillation and oscillatory translocation of Ca2+-dependent PKC.

    Uchino M, Sakai N, Kashiwagi K, Shirai Y, Shinohara Y, Hirose K, Iino M, Yamamura T and Saito N

    Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.

    Prolonged activation of metabotropic glutamate receptor 5a (mGluR5a) causes synchronized oscillations in intracellular calcium, inositol 1,4,5-trisphosphate production, and protein kinase C (PKC) activation. Additionally, mGluR5 stimulation elicited cyclical translocations of myristoylated alanine-rich protein kinase C substrate, which were opposite to that of gammaPKC (i.e. from plasma membrane to cytosol) and dependent on PKC activity, indicating that myristoylated alanine-rich protein kinase C substrate is repetitively phosphorylated by oscillating gammaPKC on the plasma membrane. Mutation of mGluR5 Thr(840) to aspartate abolished the oscillation of gammaPKC, but the mutation to alanine (T840A) did not. Cotransfection of gammaPKC with betaIIPKC, another Ca2+-dependent PKC, resulted in synchronous oscillatory translocation of both classical PKCs. In contrast, cotransfection of deltaPKC, a Ca2+-independent PKC, abolished the oscillations of both gammaPKC and inositol 1,4,5-trisphosphate. Regulation of the oscillations was dependent on deltaPKC kinase activity but not on gammaPKC. Furthermore, the T840A-mGluR5-mediated oscillations were not blocked by the deltaPKC overexpression. These results revealed that activation of mGluR5 causes translocation of both gammaPKC and deltaPKC to the plasma membrane. deltaPKC, but not gammaPKC, phosphorylates mGluR5 Thr(840), leading to the blockade of both Ca2+ oscillations and gammaPKC cycling. This subtype-specific targeting proposes the molecular basis of the multiple functions of PKC.

    The Journal of biological chemistry 2004;279;3;2254-61

  • Gene structure of the human metabotropic glutamate receptor 5 and functional analysis of its multiple promoters in neuroblastoma and astroglioma cells.

    Corti C, Clarkson RW, Crepaldi L, Sala CF, Xuereb JH and Ferraguti F

    Cambridge Brain Bank Laboratory, Department of Pathology, University of Cambridge, Level 3 Laboratory Block Addenbrooke's Hospital, Hills Road, CB2 2QQ Cambridge, United Kingdom.

    The metabotropic glutamate receptor 5 (mGluR5) has a discrete tissue expression mainly limited to neural cells. Expression of mGluR5 is developmentally regulated and undergoes dramatic changes in association with neuropathological disorders. We report the complete genomic structure of the mGluR5 gene, which is composed of 11 exons and encompasses approximately 563 kbp. Three clusters of multiple transcription initiation sites located on three distinct exons (IA, IB, and II), which undergo alternative splicing, have been identified. The 5'-flanking regions of these exons were isolated and, using a luciferase reporter gene assay, shown to possess active promoter elements in SKN-MC neuroblastoma and U178-MG astroglioma cells. Promoter IA was characterized by a CpG island; promoter IB contained a TATA box, and promoter II possessed three active Oct-1-binding sites. Preferential luciferase activity was observed in SKN-MC concomitant with differential DNA binding activity to several responsive elements, including CREB, Oct-1, C/EBP, and Brn-2. Exposure to growth factors produced enhanced expression of promoters IB and II in astroglioma cells and activation of NF-kappa B. These results suggest that alternative 5'-splicing and usage of multiple promoters may contribute regulatory mechanisms for tissue- and context-specific expression of the mGluR5 gene.

    The Journal of biological chemistry 2003;278;35;33105-19

  • Activation of metabotropic glutamate receptor mGlu5 on nuclear membranes mediates intranuclear Ca2+ changes in heterologous cell types and neurons.

    O'Malley KL, Jong YJ, Gonchar Y, Burkhalter A and Romano C

    Department of Anatomy, Washington University School of Medicine, St. Louis, Missouri 63110, USA. omalleyk@pcg.wustl.edu

    Nuclear Ca2+ plays a critical role in many cellular functions although its mode (s) of regulation is unclear. This study shows that the metabotropic glutamate receptor, mGlu5, mobilizes nuclear Ca2+ independent of cytosolic Ca2+ regulation. Immunocytochemical, ultrastructural, and subcellular fractionation techniques revealed that the metabotropic glutamate receptor, mGlu5, can be localized to nuclear membranes in heterologous cells as well as midbrain and cortical neurons. Nuclear mGlu5 receptors derived from HEK cells or cortical cell types bound [3H]quisqualate. When loaded with Oregon Green BAPTA, nuclei isolated from mGlu5-expressing HEK cells responded to the addition of glutamate with rapid, oscillatory [Ca2+] elevations that were blocked by antagonist or EGTA. In contrast, carbachol-activation of endogenous muscarinic receptors led to cytoplasmic but not nuclear Ca2+ responses. Similarly, activation of mGlu5 receptors expressed on neuronal nuclei led to sustained Ca2+ oscillatory responses. These results suggest mGlu5 may mediate intranuclear signaling pathways.

    The Journal of biological chemistry 2003;278;30;28210-9

  • Expression and functional role of mGluR3 and mGluR5 in human astrocytes and glioma cells: opposite regulation of glutamate transporter proteins.

    Aronica E, Gorter JA, Ijlst-Keizers H, Rozemuller AJ, Yankaya B, Leenstra S and Troost D

    Department of (Neuro)Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. e.aronica@amc.uva.nl

    We examined the regulation of glutamate transporter protein expression after stimulation with selective metabotropic glutamate receptor (mGluR) agonists in cultured human glial cells. mGluR3 and mGluR5 are expressed in human astrocytes and in human glioma cells in vivo as well as in vitro, as shown by either RT-PCR or western blot analysis. The selective group I agonist (S)-3,5-dihydroxyphenylglycine produced a significant down-regulation of both GLAST and GLT-1 protein expression in astrocytes cultured in the presence of growth factors. This condition mimics the morphology of reactive glial cells in vivo including an increased expression of mGluR5 protein (observed in pathological conditions). In contrast, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine, a selective agonist of group II metabotropic glutamate receptors, positively modulates the expression of GLAST and GLT-1 proteins. A similar opposite effect of (S)-3,5-dihydroxyphenylglycine and (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine was observed for the expression of EAAT3 protein in U373 glioblastoma cell line. Selective group I and II antagonists prevented these effects. Pharmacological inhibition of mitogen-activated protein kinase and phosphatidylinositol-3-K pathways reduces the induction of GLT-1 observed in response to the group II metabotropic glutamate receptor agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine. Thus, mGluR3 and mGluR5 can critically and differentially modulate the expression of glutamate transporters and may represent interesting pharmacological targets to regulate the extracellular levels of glutamate in pathological conditions.

    The European journal of neuroscience 2003;17;10;2106-18

  • Characterization of mGluR5R, a novel, metabotropic glutamate receptor 5-related gene.

    Bates B, Xie Y, Taylor N, Johnson J, Wu L, Kwak S, Blatcher M, Gulukota K and Paulsen JE

    Wyeth Research, Functional Genomics, 35 Cambridge Park Drive, Cambridge, MA 02140, USA. bbates@wyeth.com

    We report here the isolation of a novel gene termed mGluR5R (mGluR5-related). The N-terminus of mGluR5R is highly similar to the extracellular domain of metabotropic glutamate receptor 5 (mGluR5) whereas the C-terminus bears similarity to the testis-specific gene, RNF18. mGluR5R is expressed in the human CNS in a coordinate fashion with mGluR5. Although the sequence suggests that mGluR5R may be a secreted glutamate binding protein, we found that when expressed in HEK293 cells it was membrane associated and not secreted. Furthermore, mGluR5R was incapable of binding the metabotropic glutamate receptor class I selective agonist, quisqualate. Although mGluR5R could not form disulfide-mediated covalent homodimers, it was able to form a homomeric complex, presumably through noncovalent interactions. mGluR5R also formed noncovalent heteromeric associations with an engineered construct of the extracellular domain of mGluR5 as well as with full-length mGluR5 and mGluR1alpha. The ability of mGluR5R to associate with mGluR1alpha and mGluR5 suggests that it may be a modulator of class I metabotropic glutamate receptor function.

    Brain research. Molecular brain research 2002;109;1-2;18-33

  • Identification and characterization of a novel splice variant of the metabotropic glutamate receptor 5 gene in human hippocampus and cerebellum.

    Malherbe P, Kew JN, Richards JG, Knoflach F, Kratzeisen C, Zenner MT, Faull RL, Kemp JA and Mutel V

    Pharma Division, PRPN Preclinical CNS Research, Bldg 69/333, F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland. parichehr.malherbe@roche.com

    The G-protein coupled metabotropic glutamate receptor mGlu5 plays a pivotal role as a modulator of synaptic plasticity, ion channel activity and excitotoxicity. Two splice variants, hmGlu5a and -5b have been reported previously. During screening of a human brain cDNA library for hmGlu5a, we identified a novel variant (hmGlu5d) generated by alternative splicing at the C-terminal domain. The predicted hmGlu5d protein has a C-terminal 267 amino acid shorter than that of hmGlu5a. The pattern of mRNA expression of mGluR5 variants in human brain were analyzed by RT-PCR and in situ hybridization histochemistry. RT-PCR analysis demonstrated the presence of the hmGlu5d transcript, although at low level, in human whole brain, cerebellum, cerebral cortex and hippocampus. [3H]Quisqualate displayed similar affinity at the hmGlu5 splice variants (K(D) values of 80+/-8 and 54+/-17 nM for hmGlu5a and -5d receptors, respectively). For the five mGlu agonists studied, a similar rank order of potency was observed on both hmGlu5a and -5d receptors: quisqualate>glutamate>DHPG>L-CCGI approximately ACPD. MPEP inhibited the glutamate (2 microM)-induced [Ca(2+)](i) response in hmGlu5a and -5d-HEK293 cells also with similar potency (IC(50) values 25+/-1.5 and 20+/-1.4 nM, respectively). Therefore, the large truncation of the C-terminal tail of mGlu5 does not have any apparent major effect on the potency and efficacy of agonists as measured by the [Ca(2+)](i) responses or by activation of recombinant G-protein coupled inwardly rectifying K(+) (GIRK) channel currents. The only major functional difference is the increased sensitivity of hmGlu5d to protein kinase C (PKC)-mediated desensitization, relative to hmGlu5a.

    Brain research. Molecular brain research 2002;109;1-2;168-78

  • Determinants of metabotropic glutamate receptor-5-mediated Ca2+ and inositol 1,4,5-trisphosphate oscillation frequency. Receptor density versus agonist concentration.

    Nash MS, Schell MJ, Atkinson PJ, Johnston NR, Nahorski SR and Challiss RA

    Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, P. O. Box 138, University Road, Leicester, United Kingdom. msn2@le.ac.uk

    Diverse patterns of Ca(2+)(i) release differentially regulate Ca(2+)-sensitive enzymes and gene transcription, and generally the extent of agonist activation of phospholipase C-linked G protein-coupled receptors determines the type of Ca(2+) signal. We have studied global Ca(2+) oscillations arising through activation of the metabotropic glutamate receptor mGluR5a expressed in Chinese hamster ovary cells and find that these oscillations are largely insensitive to agonist concentration. Using an inducible receptor expression system and a non-competitive antagonist, in conjunction with the translocation of eGFP-PH(PLCdelta) to monitor inositol 1,4,5-trisphosphate (InsP(3)) oscillations in single cells, we show that mGluR5a density determines the frequency of these oscillations. The predominant underlying mechanism resulted from a negative feedback loop whereby protein kinase C (PKC) inhibited InsP(3) generation. Down-regulation of PKC by prolonged exposure to phorbol ester revealed a second form of Ca(2+)(i) oscillation at low agonist concentrations. These Ca(2+)(i) signals showed features typical of classic repetitive Ca(2+)-induced Ca(2+) release and were sensitive to agonist concentration. Therefore, a single receptor can stimulate two types of InsP(3)-mediated Ca(2+) signal dependent upon feedback inhibition, producing two distinct means of controlling the final pattern of Ca(2+)(i) release. Our results have physiological implications for Ca(2+) signaling in general and emphasize the importance of mGluR5 surface expression for modulating synaptic plasticity.

    The Journal of biological chemistry 2002;277;39;35947-60

  • The actin-binding protein Filamin-A interacts with the metabotropic glutamate receptor type 7.

    Enz R

    Institut für Biochemie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany. ralf.enz@biochem.uni-erlangen.de

    A yeast two-hybrid screen identified Filamin-A as a binding partner of the metabotropic glutamate receptor type 7b (mGluR7b) splice variant. In addition, Filamin-A interacted with mGluR4a, mGluR5a, mGluR5b, mGluR7a and mGluR8a. Domain mapping revealed that alternative splicing of mGluR4, mGluR7 and mGluR8 C-termini regulated the interaction. A conserved tyrosine within mGluR C-termini was identified to mediate the binding to Filamin-A. Protein interactions were verified in biochemical assays using recombinant and native proteins. Finally, co-expression of Filamin-A and mGluR7 splice variants was shown in brain regions. These findings suggest that Filamin-A may physically link metabotropic glutamate receptors to the actin cytoskeleton.

    FEBS letters 2002;514;2-3;184-8

  • Interaction between metabotropic glutamate receptor 7 and alpha tubulin.

    Saugstad JA, Yang S, Pohl J, Hall RA and Conn PJ

    Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, Oregon, USA. jsaugstad@downeurobiology.org

    Metabotropic glutamate receptors (mGluRs) mediate a variety of responses to glutamate in the central nervous system. A primary role for group-III mGluRs is to inhibit neurotransmitter release from presynaptic terminals, but the molecular mechanisms that regulate presynaptic trafficking and activity of group-III mGluRs are not well understood. Here, we describe the interaction of mGluR7, a group-III mGluR and presynaptic autoreceptor, with the cytoskeletal protein, alpha tubulin. The mGluR7 carboxy terminal (CT) region was expressed as a GST fusion protein and incubated with rat brain extract to purify potential mGluR7-interacting proteins. These studies yielded a single prominent mGluR7 CT-associated protein of 55 kDa, which subsequent microsequencing analysis revealed to be alpha tubulin. Coimmunoprecipitation assays confirmed that full-length mGluR7 and alpha tubulin interact in rat brain as well as in BHK cells stably expressing mGluR7a, a splice variant of mGluR7. In addition, protein overlay experiments showed that the CT domain of mGluR7a binds specifically to purified tubulin and calmodulin, but not to bovine serum albumin. Further pull-down studies revealed that another splice variant mGluR7b also interacts with alpha tubulin, indicating that the binding region is not localized to the splice-variant regions of either mGluR7a (900-915) or mGluR7b (900-923). Indeed, deletion mutagenesis experiments revealed that the alpha tubulin-binding site is located within amino acids 873-892 of the mGluR7 CT domain, a region known to be important for regulation of mGluR7 trafficking. Interestingly, activation of mGluR7a in cells results in an immediate and significant decrease in alpha tubulin binding. These data suggest that the mGluR7/alpha tubulin interaction may provide a mechanism to control access of the CT domain to regulatory molecules, or alternatively, that this interaction may lead to morphological changes in the presynaptic membrane in response to receptor activation.

    Funded by: NIMH NIH HHS: R01 MH059951, R01 MH059951-01; NINDS NIH HHS: NS313373, NS348876, NS36755; PHS HHS: R01-521635

    Journal of neurochemistry 2002;80;6;980-8

  • Tamalin, a PDZ domain-containing protein, links a protein complex formation of group 1 metabotropic glutamate receptors and the guanine nucleotide exchange factor cytohesins.

    Kitano J, Kimura K, Yamazaki Y, Soda T, Shigemoto R, Nakajima Y and Nakanishi S

    Department of Biological Sciences, Faculty of Medicine, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8501, Japan.

    In this investigation, we report identification and characterization of a 95 kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain-containing protein termed tamalin, also recently named GRP1-associated scaffold protein (GRASP), that interacts with group 1 metabotropic glutamate receptors (mGluRs). The yeast two-hybrid system and in vitro pull-down assays indicated that the PDZ domain-containing, amino-terminal half of tamalin directly binds to the class I PDZ-binding motif of group 1 mGluRs. The C-terminal half of tamalin also bound to cytohesins, the members of guanine nucleotide exchange factors (GEFs) specific for the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. Tamalin mRNA is expressed predominantly in the telencephalic region and highly overlaps with the expression of group 1 mGluR mRNAs. Both tamalin and cytohesin-2 were enriched and codistributed with mGluR1a in postsynaptic membrane fractions. Importantly, recombinant and native mGluR1a/tamalin/cytohesin-2 complexes were coimmunoprecipitated from transfected COS-7 cells and rat brain tissue, respectively. Transfection of tamalin and mutant tamalin lacking a cytohesin-binding domain caused an increase and decrease in cell-surface expression of mGluR1a in COS-7 cells, respectively. Furthermore, adenovirus-mediated expression of tamalin and dominant-negative tamalin facilitated and reduced the neuritic distribution of endogenous mGluR5 in cultured hippocampal neurons, respectively. The results indicate that tamalin plays a key role in the association of group 1 mGluRs with the ARF-specific GEF proteins and contributes to intracellular trafficking and the macromolecular organization of group 1 mGluRs at synapses.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;4;1280-9

  • Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homer-related, synaptic proteins.

    Xiao B, Tu JC, Petralia RS, Yuan JP, Doan A, Breder CD, Ruggiero A, Lanahan AA, Wenthold RJ and Worley PF

    Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Homer is a neuronal immediate early gene (IEG) that is enriched at excitatory synapses and binds group 1 metabotropic glutamate receptors (mGluRs). Here, we characterize a family of Homer-related proteins derived from three distinct genes. Like Homer IEG (now termed Homer 1a), all new members bind group 1 mGluRs. In contrast to Homer 1a, new members are constitutively expressed and encode a C-terminal coiled-coil (CC) domain that mediates self-multimerization. CC-Homers form natural complexes that cross-link mGluRs and are enriched at the postsynaptic density. Homer 1a does not multimerize and blocks the association of mGluRs with CC-Homer complexes. These observations support a model in which the dynamic expression of Homer 1a competes with constitutively expressed CC-Homers to modify synaptic mGluR properties.

    Funded by: NIDA NIH HHS: DA10309, DA11742; NIMH NIH HHS: KO2 MH01152; ...

    Neuron 1998;21;4;707-16

  • GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain.

    Snow BE, Hall RA, Krumins AM, Brothers GM, Bouchard D, Brothers CA, Chung S, Mangion J, Gilman AG, Lefkowitz RJ and Siderovski DP

    Amgen Institute, Toronto, Ontario M5G 2C1, Canada.

    Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein alpha subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3' end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3' end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class alpha subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3' exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.

    Funded by: NHLBI NIH HHS: HL16037; NIGMS NIH HHS: GM34497

    The Journal of biological chemistry 1998;273;28;17749-55

  • Phosphorylation and calmodulin binding of the metabotropic glutamate receptor subtype 5 (mGluR5) are antagonistic in vitro.

    Minakami R, Jinnai N and Sugiyama H

    School of Health Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan.

    Metabotropic glutamate receptors, which are members of a G protein-coupled receptor family, mediate the glutamate responses by coupling to the intracellular signal transduction pathway. We herein report that calmodulin (CaM) interacts with the metabotropic glutamate receptor subtype 5 (mGluR5) in a Ca2+-dependent manner in vitro. CaM is capable of binding on two distinct sites in the COOH-terminal intracellular region of the receptor with different affinities. The CaM binding domains are separated by an alternatively spliced exon cassette present in one of the splicing isoforms of mGluR5. By using fusion proteins and synthetic peptides we showed that protein kinase C phosphorylates both CaM binding regions. This phosphorylation is inhibited by the binding of CaM to the receptor, and conversely the binding is inhibited by the phosphorylation. These antagonisms of the CaM binding and phosphorylation thus suggest the possibility that they regulate the receptor responses in vivo.

    The Journal of biological chemistry 1997;272;32;20291-8

  • Large-scale concatenation cDNA sequencing.

    Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G and Gibbs RA

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

    Funded by: NHGRI NIH HHS: 1F32 HG00169-01, F32 HG000169, F33 HG000210, P30 HG00210-05, R01 HG00823, U54 HG003273

    Genome research 1997;7;4;353-8

  • Homer: a protein that selectively binds metabotropic glutamate receptors.

    Brakeman PR, Lanahan AA, O'Brien R, Roche K, Barnes CA, Huganir RL and Worley PF

    Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Spatial localization and clustering of membrane proteins is critical to neuronal development and synaptic plasticity. Recent studies have identified a family of proteins, the PDZ proteins, that contain modular PDZ domains and interact with synaptic ionotropic glutamate receptors and ion channels. PDZ proteins are thought to have a role in defining the cellular distribution of the proteins that interact with them. Here we report a novel dendritic protein, Homer, that contains a single, PDZ-like domain and binds specifically to the carboxy terminus of phosphoinositide-linked metabotropic glutamate receptors. Homer is highly divergent from known PDZ proteins and seems to represent a novel family. The Homer gene is also distinct from members of the PDZ family in that its expression is regulated as an immediate early gene and is dynamically responsive to physiological synaptic activity, particularly during cortical development. This dynamic transcriptional control suggests that Homer mediates a novel cellular mechanism that regulates metabotropic glutamate signalling.

    Nature 1997;386;6622;284-8

  • A "double adaptor" method for improved shotgun library construction.

    Andersson B, Wentland MA, Ricafrente JY, Liu W and Gibbs RA

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

    The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

    Funded by: NHGRI NIH HHS: R01 HG00823

    Analytical biochemistry 1996;236;1;107-13

  • Molecular and functional characterization of recombinant human metabotropic glutamate receptor subtype 5.

    Daggett LP, Sacaan AI, Akong M, Rao SP, Hess SD, Liaw C, Urrutia A, Jachec C, Ellis SB, Dreessen J et al.

    SIBIA, Inc., La Jolla, CA 92037-4641, USA.

    We have isolated and characterized overlapping cDNAs that encode two isoforms of the human metabotropic glutamate receptor subtype 5 (hmGluR5). The deduced amino acid sequences of human and rat mGluR5a are 94.5% identical. However, a region in the putative cytoplasmic domain (SER926-ALA1121) displays significant sequence divergence. Genomic analysis of this region showed that the sequence divergence results from species-specific differences in the genomic sequences, not from alternative splicing. The distribution of mGluR5 mRNA in human brain was most strongly detected throughout the hippocampus, with moderate levels in the caudate-putamen, cerebral cortex, thalamus, and deep cerebellar nuclei, and at low levels in the cerebellar cortex. Activation of both hmGluR5a and hmGluR5b transiently expressed in Xenopus oocytes and HEK293 cells was coupled to inositol phosphate (InsP) formation and elevation of the intracellular free calcium ([Ca2+]i). The agonist rank order of potency for activating recombinant hmGluR5a receptors in either system was quisqualate > L-glutamate > 1S,3R-ACPD. Both the quisqualate stimulated InsP and [Ca2+]i were inhibited by (+)-MCPG. Recombinant human mGluR5a was also stably expressed in mouse fibroblast Ltk- cells, in which the efficacy and potency of quisqualate were unchanged for more than 30 cell passages.

    Neuropharmacology 1995;34;8;871-86

  • Molecular cloning and the functional expression of two isoforms of human metabotropic glutamate receptor subtype 5.

    Minakami R, Katsuki F, Yamamoto T, Nakamura K and Sugiyama H

    Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.

    We previously reported that a variant with extra amino acids exists in rat metabotropic glutamate receptor subtype 5 (mGluR5) and that the identical extra sequence also exists in the human mGluR5 cDNA. We herein report the complete sequence and the functional expression of two isoforms of mGluR5 from the human brain. The deduced amino acid sequence of the large extracellular domain is extremely well conserved between rat and human mGluR5 (98.6%) which suggests that the amino-terminal region of mGluR5 is functionally important. We show that the glutamate-evoked responses appear in Xenopus oocytes while expressing either of the two mGluR5 isoforms, which suggests that these two receptors from the human brain could activate phospholipase C and generate Ca(2+)-activated Cl- current. We compared some of the pharmacological profiles of these two isoforms, but no clear differences could be observed. Finally, we also examined the effect of exogenous G proteins on the mGluR5-evoked responses.

    Biochemical and biophysical research communications 1994;199;3;1136-43

  • A variant of metabotropic glutamate receptor subtype 5: an evolutionally conserved insertion with no termination codon.

    Minakami R, Katsuki F and Sugiyama H

    Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.

    We herein report the presence of a variant of the rat metabotropic glutamate receptor subtype 5 (mGluR5) cDNA. In this variant, named mGluR5b, an extra 96 bp sequence is inserted into the originally reported mGluR5 cDNA. The position of this insertion corresponds to that of the 85 bp insertion reported for the alternative form of mGluR1, another subtype of the receptors (designated as mGluR1 beta). No sequence-similarity was found between the additional sequences of the mGluR1 beta and the mGluR5b. While the additional sequence of the mGluR1 beta cDNA contains a translation termination codon in the frame, that of the mGluR5b cDNA does not. This identical 96 bp insertion was also found in a human mGluR5 cDNA clone. Northern blot analyses with the mGluR5b-specific probe showed that the expression of mRNA for this species is regulated in a region-specific manner in the rat brain.

    Biochemical and biophysical research communications 1993;194;2;622-7

Gene lists (8)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000033 G2C Homo sapiens Pocklington H2 Human orthologues of cluster 2 (mouse) from Pocklington et al (2006) 13
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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