G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
glutamate receptor, ionotropic, AMPA 1
G00000013 (Mus musculus)

Databases (8)

ENSG00000155511 (Ensembl human gene)
2890 (Entrez Gene)
252 (G2Cdb plasticity & disease)
GRIA1 (GeneCards)
138248 (OMIM)
Marker Symbol
HGNC:4571 (HGNC)
Protein Expression
1965 (human protein atlas)
Protein Sequence
P42261 (UniProt)

Synonyms (1)


Literature (69)

Pubmed - other

  • C-terminal domains of transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor regulatory proteins not only facilitate trafficking but are major modulators of AMPA receptor function.

    Sager C, Terhag J, Kott S and Hollmann M

    Department of Biochemistry I-Receptor Biochemistry, Ruhr University Bochum, Germany.

    Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors are essential players in fast synaptic transmission in the vertebrate central nervous system. Their synaptic delivery and localization as well as their electrophysiological properties are regulated by transmembrane AMPA receptor regulatory proteins (TARPs). However, the exact mechanisms of how the four originally designated TARPs (gamma2, gamma3, gamma4, and gamma8) modulate AMPA receptor function remain largely unknown. Previous studies suggested the C-terminal domain (CTD) of gamma2 to mediate increased trafficking and reduced desensitization of AMPA receptors. As it remained unclear whether these findings extend to other TARPs, we set out to investigate and compare the role of the CTDs of the four original TARPs in AMPA receptor modulation. To address this issue, we replaced the TARP CTDs with the CTD of the homologous subunit gamma1, a voltage-dependent calcium channel subunit expressed in skeletal muscle that lacks TARP properties. We analyzed the impact of the resulting chimeras on GluR1 functional properties in Xenopus oocytes and HEK293 cells. Interestingly, the CTDs of all TARPs not only modulate the extent and kinetics of desensitization but also modulate agonist potencies of AMPA receptors. Furthermore, the CTDs are required for TARP-induced modulation of AMPA receptor gating, including conversion of antagonists to partial agonists and constitutive channel openings. Strikingly, we found a special role of the cytoplasmic tail of gamma4, suggesting that the underlying mechanisms of modulation of AMPA receptor function are different among the TARPs. We propose that the intracellularly located CTD is the origin of TARP-specific functional modulation and not merely a facilitator of trafficking.

    The Journal of biological chemistry 2009;284;47;32413-24

  • S-nitrosylation of stargazin regulates surface expression of AMPA-glutamate neurotransmitter receptors.

    Selvakumar B, Huganir RL and Snyder SH

    Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

    Synaptic plasticity is mediated by changes in the surface expression of AMPA receptors (AMPARs). Stargazin and related transmembrane AMPAR regulatory proteins have emerged as the principal regulators of AMPAR surface expression. Here, we show in heterologous cells and primary neurons that stargazin is physiologically S-nitrosylated, resulting in increased surface expression. S-nitrosylation of stargazin increases binding to the AMPAR subunit GluR1, causing increased surface expression of the AMPAR. NMDAR stimulation, well known to activate neuronal nitric oxide synthase, increases both nitrosylation of stargazin and its binding to AMPAR. Thus, S-nitrosylation of stargazin is a physiologic regulator of AMPAR surface expression.

    Funded by: Howard Hughes Medical Institute; NIDA NIH HHS: DA00074, K05 DA000074; NIMH NIH HHS: MH18501, R01 MH018501, R37 MH018501; PHS HHS: N536715

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;38;16440-5

  • 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

  • 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

  • Attenuated AMPA receptor expression allows glioblastoma cell survival in glutamate-rich environment.

    van Vuurden DG, Yazdani M, Bosma I, Broekhuizen AJ, Postma TJ, Heimans JJ, van der Valk P, Aronica E, Tannous BA, Würdinger T, Kaspers GJ and Cloos J

    Department of Pediatric Oncology/Hematology, VU University Medical Center, Amsterdam, The Netherlands.

    Background: Glioblastoma multiforme (GBM) cells secrete large amounts of glutamate that can trigger AMPA-type glutamate receptors (AMPARs). This commonly results in Na(+) and Ca(2+)-permeability and thereby in excitotoxic cell death of the surrounding neurons. Here we investigated how the GBM cells themselves survive in a glutamate-rich environment.

    In silico analysis of published reports shows down-regulation of all ionotropic glutamate receptors in GBM as compared to normal brain. In vitro, in all GBM samples tested, mRNA expression of AMPAR subunit GluR1, 2 and 4 was relatively low compared to adult and fetal total brain mRNA and adult cerebellum mRNA. These findings were in line with primary GBM samples, in which protein expression patterns were down-regulated as compared to the normal tissue. Furthermore, mislocalized expression of these receptors was found. Sequence analysis of GluR2 RNA in primary and established GBM cell lines showed that the GluR2 subunit was found to be partly unedited.

    Conclusions: Together with the lack of functional effect of AMPAR inhibition by NBQX our results suggest that down-regulation and afunctionality of AMPARs, enable GBM cells to survive in a high glutamate environment without going into excitotoxic cell death themselves. It can be speculated that specific AMPA receptor inhibitors may protect normal neurons against the high glutamate microenvironment of GBM tumors.

    PloS one 2009;4;6;e5953

  • AMPA receptors promote perivascular glioma invasion via beta1 integrin-dependent adhesion to the extracellular matrix.

    Piao Y, Lu L and de Groot J

    M. D. Anderson Cancer Center, Houston, TX 77030, USA.

    High-grade gliomas release excitotoxic concentrations of glutamate, which has been shown to enhance tumor proliferation and migration. alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptors are abundantly expressed at the invading edge of glioblastoma specimens, suggesting they may play an important biologic role in tumor invasion. In this study, we examined potential mechanisms by which AMPA receptor (AMPAR) expression and stimulation promote glioma cell migration and invasion. Overexpression of GluR1, the most abundant AMPAR subunit in gliomas, positively correlated with glioma cell adhesion to type I and type IV collagen, which was decreased in cells with knockdown of GluR1 and with blocking antibodies to beta1 integrin. Furthermore, stimulation of the AMPAR led to detachment of cells from the extracellular matrix (ECM). Immunoprecipitation studies showed that GluR1 associated with the actin cytoskeleton-linked protein band 4.1B (brain type), which may serve as a link between GluR1 and integrins. Overexpression of GluR1 correlated with increased cell-surface expression of beta1 integrin, increased phosphorylation of focal adhesion kinase (FAK-Y397), and enhanced numbers of focal adhesion (FA) complexes. Cells overexpressing GluR1 had increased colocalization of actin and paxillin at FAs and, in several glioma cell lines, significantly increased invasion in an in vitro Matrigel transwell assay. Likewise, in an intracranial xenograft model, overexpression of GluR1 led to perivascular and subependymal glioma cell invasion similar to patterns of tumor dissemination described in human glioblastoma. Together, these results suggest that AMPARs may link signals from the ECM to sites of FA, where signal integration promotes tumor invasion.

    Neuro-oncology 2009;11;3;260-73

  • Genetic and clinical predictors of sexual dysfunction in citalopram-treated depressed patients.

    Perlis RH, Laje G, Smoller JW, Fava M, Rush AJ and McMahon FJ

    Department of Psychiatry, Massachusetts General Hospital, Boston, MA, USA. rperlis@partners.org

    Sexual dysfunction is a major contributor to treatment discontinuation and nonadherence among patients treated with selective serotonin reuptake inhibitors (SSRIs). The mechanisms by which depressive symptoms in general, as well as SSRI exposure in particular, may worsen sexual function are not known. We examined genetic polymorphisms, including those of the serotonin and glutamate systems, for association with erectile dysfunction, anorgasmia, and decreased libido during citalopram treatment. Clinical data were drawn from a nested case-control cohort derived from the STAR(*)D study, a multicenter, prospective, effectiveness trial in outpatients with nonpsychotic major depressive disorder (MDD). Self-reports of erectile dysfunction, decreased libido, or difficulty achieving orgasm based on the Patient-Rated Inventory of Side Effects were examined among Caucasian subjects (n=1473) for whom DNA and adverse effect measures were available, and who were treated openly with citalopram for up to 14 weeks. Of 1473 participants, 799 (54%) reported decreased libido; 525 (36%) reported difficulty achieving orgasm. Of 574 men, 211 (37%) reported erectile dysfunction. Using a set-based test for association, single nucleotide polymorphisms in glutamatergic genes were associated with decreased libido (GRIA3; GRIK2), difficulty achieving orgasm (GRIA1), and difficulty achieving erection (GRIN3A) (experiment-wide permuted p<0.05 for each). Evidence of association persisted after adjustment for baseline clinical and sociodemographic differences. Likewise, evidence of association was similar when the cohort was limited to those who did not report a given adverse event at the first post-baseline visit (ie, those whose adverse events were known to be treatment emergent). These hypothesis-generating analyses suggest the potential for glutamatergic treatment targets for sexual dysfunction during major depressive episodes.

    Funded by: NIMH NIH HHS: K23 MH067060

    Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 2009;34;7;1819-28

  • AMPA receptor trafficking and synaptic plasticity require SQSTM1/p62.

    Jiang J, Parameshwaran K, Seibenhener ML, Kang MG, Suppiramaniam V, Huganir RL, Diaz-Meco MT and Wooten MW

    Department of Biological Sciences and Program in Cellular and Molecular Biosciences, Auburn University, Auburn, Alabama 36849, USA.

    SQSTM1/p62 is a multidomain/scaffold for the atypical protein kinase Cs (aPKC). Phosphorylation of AMPA receptors by PKC has been shown to regulate their insertion in the postsynaptic membrane. Here, we directly tested whether p62 could interact with AMPA receptor subunits and influence their trafficking and phosphorylation. GluR1 receptor intracellular loop L2-3 and the ZZ-type zinc finger domain of p62 are essential for the interaction between these two proteins. In this context, both p62 and aPKC-mediated phosphorylation were necessary for surface delivery of the receptor. Our findings reveal that p62 is the first protein identified that interacts with a region of the GluR receptor other than the C-terminal tail. Furthermore, mice deficient in p62 displayed impaired hippocampal CA1 long-term potentiation (LTP), along with diminished surface expression of GluR1 and phosphorylation of S818. Lastly, we identify a conserved sequence (ISExSL) shared by all p62 interacting-aPKC substrates. These findings support a model where p62 interaction and aPKC phosphorylation act together to mediate AMPA receptor trafficking and long-term synaptic plasticity in the hippocampus.

    Funded by: Howard Hughes Medical Institute; NINDS NIH HHS: NS33661, R01 NS033661, R01 NS033661-09, R01 NS036715, R01 NS036715-08

    Hippocampus 2009;19;4;392-406

  • Synaptic AMPA receptor plasticity and behavior.

    Kessels HW and Malinow R

    Department of Neuroscience, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA. hkessels@ucsd.edu

    The ability to change behavior likely depends on the selective strengthening and weakening of brain synapses. The cellular models of synaptic plasticity, long-term potentiation (LTP) and depression (LTD) of synaptic strength, can be expressed by the synaptic insertion or removal of AMPA receptors (AMPARs), respectively. We here present an overview of studies that have used animal models to show that such AMPAR trafficking underlies several experience-driven phenomena-from neuronal circuit formation to the modification of behavior. We argue that monitoring and manipulating synaptic AMPAR trafficking represents an attractive means to study cognitive function and dysfunction in animal models.

    Funded by: NIA NIH HHS: AG032132, R01 AG032132; NIMH NIH HHS: MH049159, R01 MH049159, R37 MH049159

    Neuron 2009;61;3;340-50

  • Genome-wide association study of anthropometric traits in Korcula Island, Croatia.

    Polasek O, Marusić A, Rotim K, Hayward C, Vitart V, Huffman J, Campbell S, Janković S, Boban M, Biloglav Z, Kolcić I, Krzelj V, Terzić J, Matec L, Tometić G, Nonković D, Nincević J, Pehlić M, Zedelj J, Velagić V, Juricić D, Kirac I, Belak Kovacević S, Wright AF, Campbell H and Rudan I

    Andrija Stampar School of Public Health, School of Medicine, University of Zagreb, Croatia.

    Aim: To identify genetic variants underlying six anthropometric traits: body height, body weight, body mass index, brachial circumference, waist circumference, and hip circumference, using a genome-wide association study.

    Methods: The study was carried out in the isolated population of the island of Korcula, Croatia, with 898 adult examinees who participated in the larger DNA-based genetic epidemiological study in 2007. Anthropometric measurements followed standard internationally accepted procedures. Examinees were genotyped using HumanHap 370CNV chip by Illumina, with a genome-wide scan containing 316730 single nucleotide polymorphisms (SNP).

    Results: A total of 11 SNPs were associated with the investigated traits at the level of P<10(-5), with one SNP (rs7792939 in gene zinc finger protein 498, ZNF498) associated with body weight, hip circumference, and brachial circumference (P=3.59-5.73 x 10(-6)), and another one (rs157350 in gene delta-sarcoglycan, SGCD) with both brachial and hip circumference (P=3.70-6.08 x 10(-6). Variants in CRIM1, a gene regulating delivery of bone morphogenetic proteins to the cell surface, and ITGA1, involved in the regulation of mesenchymal stem cell proliferation and cartilage production, were also associated with brachial circumference (P=7.82 and 9.68 x 10(-6), respectively) and represent interesting functional candidates. Other associations involved those between genes SEZ6L2 and MAX and waist circumference, XTP6 and brachial circumference, and AMPA1/GRIA1 and height.

    Conclusion: Although the study was underpowered for the reported associations to reach formal threshold of genome-wide significance under the assumption of independent multiple testing, the consistency of association between the 2 variants and a set of anthropometric traits makes CRIM1 and ITGA1 highly interesting for further replication and functional follow-up. Increased linkage disequilibrium between the used markers in an isolated population makes the formal significance threshold overly stringent, and changed allele frequencies in isolate population may contribute to identifying variants that would not be easily identified in large outbred populations.

    Funded by: Medical Research Council: MC_U127561128

    Croatian medical journal 2009;50;1;7-16

  • Polymorphisms in the GRIA1 gene region in psychotic bipolar disorder.

    Kerner B, Jasinska AJ, DeYoung J, Almonte M, Choi OW and Freimer NB

    Center for Neurobehavioral Genetics, Department of Psychiatry and Biobehavioral Sciences, Semel Institute for Neuroscience and Human Behavior, University of California at Los Angeles, 90095-1761, USA. bkerner@mednet.ucla.edu

    We reported previously a significant linkage signal between psychotic bipolar disorder (BP) and microsatellite markers on chromosome 5q31-34 in the National Institute of Mental Health Bipolar Genetics Initiative (NIMH-BPGI) data set, Wave 1. In an attempt to fine-map this linkage signal we genotyped 1,134 single nucleotide polymorphisms (SNPs) under the linkage peak in 23 informative families (131 individuals) with evidence of linkage. We tested family based association in the presence of linkage with the computer software package FBAT. The most significant association in these families was with a SNP in the second intron of GRIA1 (alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) subunit 1 receptor gene) (rs490922, Z-score = 3.3, P = 0.001). The analysis of 37 additional families with psychotic BP from NIMH-BPGI data sets, Waves 2, 3, and 4 revealed a signal at a SNP in intron 5 of the GRIA1 gene (rs4385264, Z-score = 3.2, P-value = 0.002). A combined analysis of all 60 families continued to support evidence for association of GRIA1 with psychotic BP; however, individual SNPs could not be replicated across datasets. The AMPA1 receptor has been shown to influence cognitive function, such as working memory and reward learning. Our findings suggest that variations in this receptor may contribute to the pathophysiology of BP with psychotic features in some families.

    Funded by: NIMH NIH HHS: K08 MH074057, K08 MH074057-03, KO2 MH001374, KO8 MH074057, R01 MH049499, R01 MH059533, R01 MH059534, R01 MH059535, R01 MH059545, R01 MH059548, R01 MH059553, R01 MH059556, R01 MH059567, R01 MH060068, Z01 MH002810

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;1;24-32

  • Neurotransmission and bipolar disorder: a systematic family-based association study.

    Shi J, Badner JA, Hattori E, Potash JB, Willour VL, McMahon FJ, Gershon ES and Liu C

    Department of Psychiatry, University of Chicago, Chicago, Illinois 60637, USA. kjacobso@bsd.uchicago.edu

    Neurotransmission pathways/systems have been proposed to be involved in the pathophysiology and treatment of bipolar disorder for over 40 years. In order to test the hypothesis that common variants of genes in one or more of five neurotransmission systems confer risk for bipolar disorder, we analyzed 1,005 tag single nucleotide polymorphisms in 90 genes from dopaminergic, serotonergic, noradrenergic, GABAergic, and glutamatergic neurotransmitter systems in 101 trios and 203 quads from Caucasian bipolar families. Our sample has 80% power to detect ORs >or= 1.82 and >or=1.57 for minor allele frequencies of 0.1 and 0.5, respectively. Nominally significant allelic and haplotypic associations were found for genes from each neurotransmission system, with several reaching gene-wide significance (allelic: GRIA1, GRIN2D, and QDPR; haplotypic: GRIN2C, QDPR, and SLC6A3). However, none of these associations survived correction for multiple testing in an individual system, or in all systems considered together. Significant single nucleotide polymorphism associations were not found with sub-phenotypes (alcoholism, psychosis, substance abuse, and suicide attempts) or significant gene-gene interactions. These results suggest that, within the detectable odds ratios of this study, common variants of the selected genes in the five neurotransmission systems do not play major roles in influencing the risk for bipolar disorder or comorbid sub-phenotypes.

    Funded by: NIAAA NIH HHS: AA00231, U10AA0503; NIDA NIH HHS: 5R01DA018662; NIMH NIH HHS: R01 MH061613, R01 MH061613-08, R01 MH065560, R01 MH065560-05, R01 MH080425, R01 MH080425-01

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147B;7;1270-7

  • Knockdown of GluR1 expression by RNA interference inhibits glioma proliferation.

    de Groot JF, Piao Y, Lu L, Fuller GN and Yung WK

    Brain Tumor Center, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA. jdegroot@mdanderson.org

    High-grade gliomas release excitotoxic concentrations of glutamate which contributes to their malignant phenotype. To improve our understanding of the mechanisms by which glutamate enhances tumor growth and invasion, we examined alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-mediated signaling in glioma cell lines. shRNA was used to stably knockdown GluR1, the most abundant AMPA receptor subunit in glioma, to evaluate its role in tumor signaling, proliferation and tumorigenicity. In a tissue array, there was a statistically significant increase in GluR1 expression in glioblastoma samples compared to anaplastic astrocytoma and low-grade tumors. In vitro, we observed a time and dose-dependent increase in MAPK phosphorylation following exposure to AMPA, which was blocked with AMPA receptor antagonists and the MEK1 inhibitor PD98059. Retroviral delivery of GluR1 shRNA in U251 and U87 glioma cells reduced GluR1 protein expression, inhibited AMPA-mediated increases in MAPK phosphorylation, and decreased glioma proliferation in vitro. U251 and U87 shGluR1 cells implanted into the flanks of nude mice grew slower than controls, which correlated with a decrease in proliferation measured by Ki-67 staining and an increase in apoptosis. These results suggest that AMPA receptors are abundantly expressed in high-grade gliomas and gene silencing of the GluR1 AMPA receptor subunit results in abrogation of AMPA-mediated signaling and tumor growth.

    Journal of neuro-oncology 2008;88;2;121-33

  • AKAP79 selectively enhances protein kinase C regulation of GluR1 at a Ca2+-calmodulin-dependent protein kinase II/protein kinase C site.

    Tavalin SJ

    Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. stavalin@utmem.edu

    Enhancement of AMPA receptor activity in response to synaptic plasticity inducing stimuli may arise, in part, through phosphorylation of the GluR1 AMPA receptor subunit at Ser-831. This site is a substrate for both Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). However, neuronal protein levels of CaMKII may exceed those of PKC by an order of magnitude. Thus, it is unclear how PKC could effectively regulate this common target site. The multivalent neuronal scaffold A-kinase-anchoring protein 79 (AKAP79) is known to bind PKC and is linked to GluR1 by synapse-associated protein 97 (SAP97). Here, biochemical studies demonstrate that AKAP79 localizes PKC activity near the receptor, thus accelerating Ser-831 phosphorylation. Complementary electrophysiological studies indicate that AKAP79 selectively shifts the dose-dependence for PKC modulation of GluR1 receptor currents approximately 20-fold, such that low concentrations of PKC are as effective as much higher CaMKII concentrations. By boosting PKC activity near a target substrate, AKAP79 provides a mechanism to overcome limitations in kinase abundance thereby ensuring faithful signal propagation and efficient modification of AMPA receptor-mediated responses.

    Funded by: NINDS NIH HHS: NS46661, R01 NS046661

    The Journal of biological chemistry 2008;283;17;11445-52

  • Motor protein-dependent transport of AMPA receptors into spines during long-term potentiation.

    Correia SS, Bassani S, Brown TC, Lisé MF, Backos DS, El-Husseini A, Passafaro M and Esteban JA

    Department of Pharmacology, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109-0632, USA.

    The regulated trafficking of neurotransmitter receptors at synapses is critical for synaptic function and plasticity. However, the molecular machinery that controls active transport of receptors into synapses is largely unknown. We found that, in rat hippocampus, the insertion of AMPA receptors (AMPARs) into spines during synaptic plasticity requires a specific motor protein, which we identified as myosin Va. We found that myosin Va associates with AMPARs through its cargo binding domain. This interaction was enhanced by active, GTP-bound Rab11, which is also transported by the motor protein. Myosin Va mediated the CaMKII-triggered translocation of GluR1 receptors from the dendritic shaft into spines, but it was not required for constitutive GluR2 trafficking. Accordingly, myosin Va was specifically required for long-term potentiation, but not for basal synaptic transmission. In summary, we identified the specific motor protein and organelle acceptor that catalyze the directional transport of AMPARs into spines during activity-dependent synaptic plasticity.

    Funded by: NIMH NIH HHS: F31-MH070205, MH070417; Telethon: TCR07006

    Nature neuroscience 2008;11;4;457-66

  • Receptor occupancy and channel-opening kinetics: a study of GLUR1 L497Y AMPA receptor.

    Pei W, Ritz M, McCarthy M, Huang Z and Niu L

    Department of Chemistry and Center for Neuroscience Research, University at Albany, State University of New York, New York 12222, USA.

    AMPA glutamate ion channels are tetrameric receptors in which activation to form the open channel depends on the binding of possibly multiple glutamate molecules. However, it is unclear whether AMPA receptors bound with a different number of glutamate molecules (i.e. one being the minimal and four being the maximal number of glutamate molecules) open the channels with different kinetic constants. Using a laser pulse photolysis technique that provides microsecond time resolution, we investigated the channel-opening kinetic mechanism of a nondesensitizing AMPA receptor, i.e. GluR1Q(flip) L497Y or a leucine-to-tyrosine substitution mutant, in the entire range of glutamate concentrations to ensure receptor saturation. We found that the minimal number of glutamate molecules required to bind to the receptor and to open the channel is two (or n = 2), and that the entire channel-opening kinetics can be adequately described by just one channel-opening rate constant, k(op), which correlates to n = 2. This result suggests that higher receptor occupancy (n = 3 and 4) does not give rise to different k(op) values or, at least, not appreciably if the k(op) values are different. Furthermore, compared with the wild-type receptor (Li, G., and Niu, L. (2004) J. Biol. Chem. 279, 3990-3997), the channel-opening and channel-closing rate constants of the mutant are 1.5- and 13-fold smaller, respectively. Thus, the major effect of this mutation is to decrease the channel-closing rate constant by stabilizing the open channel conformation.

    The Journal of biological chemistry 2007;282;31;22731-6

  • Stargazin interaction with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors is critically dependent on the amino acid at the narrow constriction of the ion channel.

    Körber C, Werner M, Hoffmann J, Sager C, Tietze M, Schmid SM, Kott S and Hollmann M

    Department of Biochemistry I-Receptor Biochemistry, Ruhr University Bochum, D-44780 Bochum, Germany.

    The subunit GluR2 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subfamily of ionotropic glutamate receptors (GluRs) features a single amino acid at the narrow constriction of the pore loop that is altered from glutamine to arginine by RNA editing. This so-called Q/R site has been shown to play an important role in the determination of the electrophysiological properties of AMPA receptor complexes as well as of trafficking to the plasma membrane. The protein stargazin has also been shown to modulate electrophysiological properties and trafficking to the plasma membrane of AMPA receptors. In this study we examined via a series of mutants of the Q/R site of the AMPA receptor GluR1 whether the amino acid at this position has any influence on the modulatory effects mediated by stargazin. To this end, we analyzed current responses of Q/R site mutants upon application of glutamate and kainate and determined the amount of mutant receptor protein in the plasma membrane in Xenopus oocytes. Desensitization kinetics of several mutants were analyzed in HEK293 cells. We found that the stargazin-mediated decrease in receptor desensitization, the slowing of desensitization kinetics, and the kainate efficacy were all dependent on the amino acid at the Q/R site, whereas the stargazin-mediated increase in trafficking toward the plasma membrane remained independent of this amino acid. We propose that the Q/R site modulates the interaction of stargazin with the transmembrane domains of AMPA receptors via an allosteric mechanism and that this modulation leads to the observed differences in the electrophysiological properties of the receptor.

    The Journal of biological chemistry 2007;282;26;18758-66

  • Electrophysiological properties of AMPA receptors are differentially modulated depending on the associated member of the TARP family.

    Kott S, Werner M, Körber C and Hollmann M

    Department of Biochemistry I, Receptor Biochemistry, Ruhr University Bochum, D-44780 Bochum, Germany.

    The family of AMPA receptors is encoded by four genes that are differentially spliced to result in the flip or flop versions of the four subunits GluR1 to GluR4. GluR2 is further modified at the so-called Q/R site by posttranscriptional RNA editing. Delivery of AMPA receptors to the plasma membrane and synaptic trafficking are controlled by transmembrane AMPA receptor regulatory proteins (TARPs). Additionally, TARPs influence essential electrophysiological properties of AMPA receptor channels such as desensitization and agonist efficacies. Here, we compare the influence of all known TARPs (gamma2, gamma3, gamma4, and gamma8) on agonist-induced currents of the four AMPA receptor subunits, including flip and flop splice variants and editing variants. We show that, although agonist-induced currents of all homomeric AMPA receptor subunits as well as all heteromeric combinations tested are significantly potentiated when coexpressed with members of the TARP family in Xenopus laevis oocytes, the extent of TARP-mediated increase in agonist-induced responses is highly dependent on both the AMPA receptor subunit and the coexpressed TARP. Moreover, we demonstrate that the splice variant of the AMPA receptor plays a key role in determining the modulation of electrophysiological properties by associated TARPs. We furthermore present evidence that individual TARP-AMPA receptor interactions control the degree of desensitization of AMPA receptors. Consequently, because of their subunit-specific impact on the electrophysiological properties, TARPs play a major role as modulatory subunits of AMPA receptors and thus contribute to the functional diversity of AMPA receptors encountered in the CNS.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;14;3780-9

  • Targeting AMPA receptor gating processes with allosteric modulators and mutations.

    Mitchell NA and Fleck MW

    Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York 12208, USA.

    Allosteric modulators and mutations that slow AMPAR desensitization have additional effects on deactivation and agonist potency. We investigated whether these are independent actions or the natural consequence of slowing desensitization. Effects of cyclothiazide (CTZ), trichlormethiazide (TCM), and CX614 were compared at wild-type GluR1 and "nondesensitizing" GluR1-L497Y mutant receptors by patch-clamp recording with ultrafast perfusion. CTZ, TCM, or L/Y mutation all essentially blocked GluR1 desensitization; however, the effects of L/Y mutation on deactivation and glutamate EC50 were three to five times greater than for modulators. CTZ and TCM further slowed desensitization of L/Y mutant receptors but paradoxically accelerated deactivation and increased agonist EC50. Results indicate that CTZ and TCM target deactivation and agonist potency independently of desensitization, most likely by modifying agonist dissociation (koff). Conversely, CX614 slowed desensitization and deactivation without affecting EC50 in both wild-type and L/Y receptors. The S750Q or combined L497Y-S750Q mutations abolished all CTZ and TCM actions without disrupting CX614 activity. Notably, the S/Q mutation also restored L/Y deactivation and EC50 to wild-type levels without restoring desensitization, further demonstrating that desensitization can be modulated independently of deactivation and EC50 by mutagenesis and possibly by allosteric modulators.

    Funded by: NINDS NIH HHS: NS040347, R01 NS040347

    Biophysical journal 2007;92;7;2392-402

  • Glutamate AMPA receptor subunit 1 gene (GRIA1) and DSM-IV-TR schizophrenia: a pilot case-control association study in an Italian sample.

    Magri C, Gardella R, Barlati SD, Podavini D, Iatropoulos P, Bonomi S, Valsecchi P, Sacchetti E and Barlati S

    Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, Brescia University School of Medicine, Brescia, Italy.

    Glutamatergic dysfunction is one of the major hypotheses for the pathogenesis of schizophrenia. The GRIA1 gene encodes for one (GluR1) of the four (GluR1-4) ionotropic AMPA receptor subunits. GRIA1 is a good candidate gene for susceptibility to schizophrenia since it maps in 5q33, a region where the presence of susceptibility loci has been suggested by independent genome-wide scans and because its expression has been found to be decreased in the brain of some schizophrenia patients. We present data from a case-control association study on the Italian population with eight polymorphisms spanning the whole GRIA1 gene. Single-locus analysis revealed a significantly different allele distribution in cases and in controls of two SNPs (rs707176, 0.41 vs. 0.31, P = 0.009; rs2963944, 0.41 vs. 0.30, P = 0.007), and one microsatellite (rs10631988, allele 9: 0.40 vs. 0.29, P = 0.004). Haplotype analysis showed an increased frequency of a specific haplotype for these markers (C09CC, 0.39 vs. 0.28, P = 0.009). Therefore our data indicate that GRIA1 may be involved in susceptibility to DSM-IV-TR schizophrenia.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2006;141B;3;287-93

  • PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking.

    Lu W and Ziff EB

    Program in Neuroscience and Physiology, New York University School of Medicine, New York, New York 10016, USA.

    PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.

    Funded by: NIMH NIH HHS: MH067229

    Neuron 2005;47;3;407-21

  • C-terminal truncation affects kinetic properties of GluR1 receptors.

    Suzuki E, Kessler M and Arai AC

    Department of Pharmacology, Southern Illinois University School of Medicine, MC 9629, 801 N. Rutledge, Room 3275, Springfield, IL 62702, USA.

    GluR1flop receptors in which the C-terminal 52 amino acids had been recombinantly removed were characterized with whole-cell recording and binding assays. Compared to wildtype GluR1, truncated receptors showed faster desensitization and deactivation and they recovered more slowly from desensitization. The EC50 for glutamate was increased 2-fold. In binding tests, K(D)s for [3H]fluorowillardiine were 1.5 times larger for truncated receptors. According to receptor simulations, most differences can be explained if the C-terminal domain is assumed to stabilize the ligand-bound closed and open states. The effects on response waveforms are different from those caused by phosphorylation, suggesting that the C-terminus influences receptor function in multiple ways. Truncated forms of GluR1 identical or similar to the one examined here may also be generated by calcium-activated proteases during intense synaptic activity. The lowered affinity and faster inactivation of these receptors suggests that their presence does not represent a risk for neuronal viability.

    Funded by: NINDS NIH HHS: NS41020

    Molecular and cellular neurosciences 2005;29;1;1-10

  • Protein-protein coupling/uncoupling enables dopamine D2 receptor regulation of AMPA receptor-mediated excitotoxicity.

    Zou S, Li L, Pei L, Vukusic B, Van Tol HH, Lee FJ, Wan Q and Liu F

    Department of Neuroscience, Centre for Addiction and Mental Health, University of Toronto, Toronto, Ontario, Canada M5T 1R8.

    here is considerable evidence that dopamine D2 receptors can modulate AMPA receptor-mediated neurotoxicity. However, the molecular mechanism underlying this process remains essentially unclear. Here we report that D2 receptors inhibit AMPA-mediated neurotoxicity through two pathways: the activation of phosphoinositide-3 kinase (PI-3K) and downregulation of AMPA receptor plasma membrane expression, both involving a series of protein-protein coupling/uncoupling events. Agonist stimulation of D2 receptors promotes the formation of the direct protein-protein interaction between the third intracellular loop of the D2 receptor and the ATPase N-ethylmaleimide-sensitive factor (NSF) while uncoupling the NSF interaction with the carboxyl tail (CT) of the glutamate receptor GluR2 subunit of AMPA receptors. Previous studies have shown that full-length NSF directly couples to the GluR2CT and facilitates AMPA receptor plasma membrane expression. Furthermore, the CT region of GluR2 subunit is also responsible for several other intracellular protein couplings, including p85 subunit of PI-3K. Therefore, the direct coupling of D2-NSF and concomitant decrease in the NSF-GluR2 interaction results in a decrease of AMPA receptor membrane expression and an increase in the interaction between GluR2 and the p85 and subsequent activation of PI-3K. Disruption of the D2-NSF interaction abolished the ability of D2 receptor to attenuate AMPA-mediated neurotoxicity by blocking the D2 activation-induced changes in PI-3K activity and AMPA receptor plasma membrane expression. Furthermore, the D2-NSF-GluR2-p85 interactions are also responsible for the D2 inhibition of ischemia-induced cell death. These data may provide a new avenue to identify specific targets for therapeutics to modulate glutamate receptor-governed diseases, such as stroke.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;17;4385-95

  • A novel scaffold protein, TANC, possibly a rat homolog of Drosophila rolling pebbles (rols), forms a multiprotein complex with various postsynaptic density proteins.

    Suzuki T, Li W, Zhang JP, Tian QB, Sakagami H, Usuda N, Usada N, Kondo H, Fujii T and Endo S

    Department of Neuroplasticity, Institute on Ageing and Adaptation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. suzukit@sch.md.shinshu-u.ac.jp

    We cloned from the rat brain a novel gene, tanc (GenBank Accession No. AB098072), which encoded a protein containing three tetratricopeptide repeats (TPRs), ten ankyrin repeats and a coiled-coil region, and is possibly a rat homolog of Drosophila rolling pebbles (rols). The tanc gene was expressed widely in the adult rat brain. Subcellular distribution, immunohistochemical study of the brain and immunocytochemical studies of cultured neuronal cells indicated the postsynaptic localization of TANC protein of 200 kDa. Pull-down experiments showed that TANC protein bound PSD-95, SAP97, and Homer via its C-terminal PDZ-binding motif, -ESNV, and fodrin via both its ankyrin repeats and the TPRs together with the coiled-coil domain. TANC also bound the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. An immunoprecipitation study showed TANC association with various postsynaptic proteins, including guanylate kinase-associated protein (GKAP), alpha-internexin, and N-methyl-D-aspartate (NMDA)-type glutamate receptor 2B and AMPA-type glutamate receptor (GluR1) subunits. These results suggest that TANC protein may work as a postsynaptic scaffold component by forming a multiprotein complex with various postsynaptic density proteins.

    The European journal of neuroscience 2005;21;2;339-50

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Differential preservation of AMPA receptor subunits in the hippocampi of Alzheimer's disease patients according to Braak stage.

    Carter TL, Rissman RA, Mishizen-Eberz AJ, Wolfe BB, Hamilton RL, Gandy S and Armstrong DM

    Laboratory of Neuronal Vulnerability and Aging, The Lankenau Institute for Medical Research, Jefferson Health System, Wynewood, PA 19096, USA. Troy.Carter@jefferson.edu

    The Alzheimer's disease (AD) brain, characterized pathologically by the presence of senile plaques and neurofibrillary tangles, contains regions that are differentially prone toward development of AD pathology. Within these "vulnerable" regions, specific cell populations appear to be selectively affected; the pyramidal cells of the hippocampal subiculum subfield constitute such a vulnerable region. This study investigated whether the AMPA receptor subunit content (GluR1, GluR2, GluR2/3) within "vulnerable" vs. "resistant" sectors of the hippocampus is quantitatively altered with increasing AD neuropathology, as determined by Braak staging. We hypothesize that the glutamate-mediated vulnerability is highly influenced by the repertoire of glutamate receptors expressed on hippocampal neurons. Our results indicate that AMPA receptor subunit proteins are relatively spared across all Braak stages in resistant subfields (CA2/CA3/Dentate Gyrus). However, within vulnerable sectors, i.e., subiculum, GluR2, and GluR2/3 protein levels decreased 63.77% and 60.60%, respectively, in association with Braak stages I-II and stages III-IV, respectively. In Braak stages V-VI, GluR2 and GluR2/3 protein levels were similar to those of Braak stages I-II. In contrast to GluR2 and GluR2/3, GluR1 protein levels were unchanged within vulnerable sectors throughout all stages of the disease. In interpreting these data, it may be relevant to consider that the GluR2 subunit impedes the flow of Ca(+2) through the AMPA receptor ion channel. Thus, we hypothesize that in resistant sectors, the presence of the GluR2 subunit may provide a neuroprotective role by limiting the flow of extracellular Ca(+2), whereas in vulnerable regions, the reduction of GluR2 may contribute to the vulnerability via a mechanism involving an increase in intracellular Ca(+2) and destabilization of intracellular Ca(+2) homeostasis.

    Funded by: NIA NIH HHS: AG08206, AG10491; NINDS NIH HHS: NS41017

    Experimental neurology 2004;187;2;299-309

  • How fast does the GluR1Qflip channel open?

    Li G and Niu L

    Department of Chemistry and the Center for Neuroscience Research, State University of New York, Albany, New York 12222, USA.

    Opening of a ligand-gated ion channel is the step at which the binding of a neurotransmitter is transduced into the electrical signal by allowing ions to flow through the transmembrane channel, thereby altering the postsynaptic membrane potential. We report the kinetics for the opening of the GluR1Qflip channel, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit of the ionotropic glutamate receptors. Using a laser-pulse photolysis technique that permits glutamate to be liberated photolytically from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (caged glutamate) with a time constant of approximately 30 micros, we show that, after the binding of glutamate, the channel opened with a rate constant of (2.9 +/- 0.2) x 10(4) s(-1) and closed with a rate constant of (2.1 +/- 0.1) x 10(3) s(-1). The observed shortest rise time (20-80% of the receptor current response), i.e. the fastest time by which the GluR1Qflip channel can open, was predicted to be 35 micros. This value is three times shorter than those previously reported. The minimal kinetic mechanism for channel opening consists of binding of two glutamate molecules, with the channel-opening probability being 0.93 +/- 0.10. These findings identify GluR1Qflip as one of the temporally efficient receptors that transduce the binding of chemical signals (i.e. glutamate) into an electrical impulse.

    The Journal of biological chemistry 2004;279;6;3990-7

  • AMPA receptor alterations precede mossy fiber sprouting in young children with temporal lobe epilepsy.

    Lynd-Balta E, Pilcher WH and Joseph SA

    Department of Neurosurgery, University of Rochester Medical Center, Rochester, NY 14642, USA.

    Following neurological injury early in life numerous events, including excitotoxicity, neural degeneration, gliosis, neosynaptogenesis, and circuitry reorganization, may alone or in concert contribute to hyperexcitability and recurrent seizures in temporal lobe epilepsy. Our studies provide new evidence regarding the temporal sequence of key elements of hippocampal reorganization, mossy fiber sprouting and glutamate receptor subunit up-regulation, in a subset of young temporal lobe epileptic patients. Without evidence of mossy fiber sprouting, the youngest age group (3-10 years old) of mesial temporal lobe epileptic patients demonstrated enhanced glutamate receptor subunit profiles, suggesting that the dendritic change precedes axonal sprouting. However, sclerotic hippocampal specimens from epileptic patients ages 12-15 years old had the characteristic features of glutamate receptor up-regulation and mossy fiber sprouting first identified in the adult, indicating that reconstructed circuits appear early in the course of the disease. Non-sclerotic hippocampal specimens from lesion associated temporal lobe epileptic patients of all age groups showed minimal cell loss, sparse staining of glutamate receptor subunits in the dentate gyrus, and little or no mossy fiber sprouting. These compelling findings suggest a progressive sequence of events in the reorganization of the dentate gyrus of sclerotic hippocampal specimens. We suggest that cell loss and up-regulation of glutamate receptor subunits appear early in temporal lobe epilepsy and contribute to the synaptic plasticity that may facilitate the subsequent sprouting of mossy fiber collaterals which compound an already precipitous state of decline. The combination of pre-synaptic and post-synaptic changes serves as a potential substrate for hyperexcitability.

    Neuroscience 2004;126;1;105-14

  • Activation of the TRPC1 cation channel by metabotropic glutamate receptor mGluR1.

    Kim SJ, Kim YS, Yuan JP, Petralia RS, Worley PF and Linden DJ

    Department of Neuroscience, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, Maryland 21205, USA.

    Group I metabotropic glutamate receptors (consisting of mGluR1 and mGluR5) are G-protein-coupled neurotransmitter receptors that are found in the perisynaptic region of the postsynaptic membrane. These receptors are not activated by single synaptic volleys but rather require bursts of activity. They are implicated in many forms of neural plasticity including hippocampal long-term potentiation and depression, cerebellar long-term depression, associative learning, and cocaine addiction. When activated, group I mGluRs engage two G-protein-dependent signalling mechanisms: stimulation of phospholipase C and activation of an unidentified, mixed-cation excitatory postsynaptic conductance (EPSC), displaying slow activation, in the plasma membrane. Here we report that the mGluR1-evoked slow EPSC is mediated by the TRPC1 cation channel. TRPC1 is expressed in perisynaptic regions of the cerebellar parallel fibre-Purkinje cell synapse and is physically associated with mGluR1. Manipulations that interfere with TRPC1 block the mGluR1-evoked slow EPSC in Purkinje cells; however, fast transmission mediated by AMPA-type glutamate receptors remains unaffected. Furthermore, co-expression of mGluR1 and TRPC1 in a heterologous system reconstituted a mGluR1-evoked conductance that closely resembles the slow EPSC in Purkinje cells.

    Nature 2003;426;6964;285-91

  • CaMKII-dependent phosphorylation regulates SAP97/NR2A interaction.

    Gardoni F, Mauceri D, Fiorentini C, Bellone C, Missale C, Cattabeni F and Di Luca M

    Center of Excellence on Neurodegenerative Diseases and Department of Pharmacological Sciences, University of Milano, via Balzaretti 9, 20133 Milano, Italy. Fabrizio.Gardoni@unimi.it

    Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and alphaCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-D-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro "pull-out" assay and in co-immunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.

    The Journal of biological chemistry 2003;278;45;44745-52

  • Ligand-independent CXCR2 dimerization.

    Trettel F, Di Bartolomeo S, Lauro C, Catalano M, Ciotti MT and Limatola C

    Dipartimento di Fisiologia Umana e Farmacologia, Università di Roma La Sapienza, Piazzale Aldo Moro 5, Rome 00185.

    Homo- and hetero-oligomerization have been reported for several G protein-coupled receptors (GPCRs). The CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth-related gene product beta) to induce cell chemotaxis. We have investigated the oligomerization of CXCR2 receptors expressed in human embryonic kidney cells and generated a series of truncated mutants to determine whether they could negatively regulate the wild-type (wt) receptor functions. CXCR2 receptor oligomerization was also studied by coimmunoprecipitation of green fluorescent protein- and V5-tagged CXCR2. Truncated CXCR2 receptors retained their ability to form oligomers only if the region between the amino acids Ala-106 and Lys-163 was present. In contrast, all of the deletion mutants analyzed were able to form heterodimers with the wt CXCR2 receptor, albeit with different efficiency, competing for wt/wt dimer formation. The truncated CXCR2 mutants were not functional and, when coexpressed with wt CXCR2, interfered with receptor functions, impairing cell signaling and chemotaxis. When CXCR2 was expressed with the AMPA-type glutamate receptor GluR1, CXCR2 dimerization was again impaired in a dose-dependent way, and receptor functions were prejudiced. In contrast, CXCR1, a chemokine receptor that shares many similarities with CXCR2, did not dimerize alone or with CXCR2 and when coexpressed with CXCR2 did not impair receptor signaling and chemotaxis. The formation of CXCR2 dimers was also confirmed in cerebellar neuron cells. Taken together, we conclude from these studies that CXCR2 functions as a dimer and that truncated receptors negatively modulate receptor activities competing for the formation of wt/wt dimers.

    Funded by: Telethon: E.0912

    The Journal of biological chemistry 2003;278;42;40980-8

  • Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain.

    Pasternack A, Coleman SK, Féthière J, Madden DR, LeCaer JP, Rossier J, Pasternack M and Keinänen K

    Viikki Biocenter, Department of Biosciences (Division of Biochemistry), Viikinkaari 5D, FIN-00014 University of Helsinki, Finland.

    The ligand-binding domains of AMPA receptor subunits carry two conserved N-glycosylation sites. In order to gain insight into the functional role of the corresponding N-glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.

    Journal of neurochemistry 2003;84;5;1184-92

  • Silence analysis of AMPA receptor mutated at the CaM-kinase II phosphorylation site.

    Derkach VA

    Vollum Institute, Oregon Health Sciences University, Portland 97201, USA. derkachv@ohsu.edu

    Direct phosphorylation of the GluR1 subunit of postsynaptic AMPA receptors by Ca(2+)/calmodulin-dependent protein kinase II (CaM-KII) is believed to be one of the major contributors to the enhanced strength of glutamatergic synapses in CA1 area of hippocampus during long-term potentiation. The molecular mechanism of AMPA receptor regulation by CaM-KII is examined here by a novel approach, silence analysis, which is independent of previously used variance analysis. I show that three fundamental channel properties-single-channel conductance, channel open probability, and the number of functional channels-can be measured in an alternative way, by analyzing the probability of channels to be simultaneously closed (silent). Validity of the approach was confirmed by modeling, and silence analysis was applied then to the GluR1 AMPA receptor mutated at S831, the site phosphorylated by CaM-KII during long-term potentiation. Silence analysis indicates that a negative charge at S831 is a critical determinant for the enhanced channel function as a charge carrier. Silence and variance analyses, when applied to the same sets of data, were in agreement on the receptor regulation upon mutations. These results provide independent evidences for the mechanism of AMPA receptor regulation by CaM-KII and further strengthens the idea how calcium-dependent phosphorylation of AMPA receptors can contribute to the plasticity at central glutamatergic synapses.

    Biophysical journal 2003;84;3;1701-8

  • Heteromer formation of delta2 glutamate receptors with AMPA or kainate receptors.

    Kohda K, Kamiya Y, Matsuda S, Kato K, Umemori H and Yuzaki M

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA.

    The delta2 glutamate receptor (GluRdelta2) is predominantly expressed in the postsynaptic densities of parallel fiber-Purkinje cell synapses and plays a crucial role in cerebellar function. However, the mechanisms by which GluRdelta2 participates in cerebellar functions are largely unknown because GluRdelta2 does not bind glutamate analogs. We investigated the possibility that GluRdelta2 may be involved in channel formation together with other glutamate receptor families. We transiently expressed lurcher mutant AMPA receptor GluR1(Lc) and kainate receptor GluR6(Lc) in HEK293 cells. Cells expressing these constitutively active channels displayed a rectifying current-voltage (I-V) relationship. However, when cells were co-transfected with GluRdelta2(Lc), which had the arginine residue in the channel pore region, cells displayed a linear I-V relationship, a result that indicates GluRdelta2(Lc) formed functional heteromeric channels with GluR1(Lc) or GluR6(Lc). Assembly of GluRdelta2 with GluR1 or GluR6 was further confirmed by co-immunoprecipitation assays in HEK293 cells. In addition, GluRdelta2 receptors were partially co-immunoprecipitated from cerebellar synaptosomal fractions by antibodies against GluR2 or KA2. In contrast to lurcher channels, expression of wild-type GluRdelta2 significantly reduced the glutamate-induced current of the wild-type GluR1 receptors without affecting channel properties, such as current kinetics, dose-response relationship, and single-channel conductance. Thus, the heteromeric channel created by the association of wild-type GluR1 and GluRdelta2 may not be gated by glutamate and does not participate in glutamate-induced currents. These results suggest that GluRdelta2 and AMPA or kainate receptors can assemble to form heteromeric receptors in vitro and could modify glutamate signaling in vivo. These findings may help explain the role of GluRdelta2.

    Funded by: NCI NIH HHS: CA 21765; NINDS NIH HHS: NS 36925

    Brain research. Molecular brain research 2003;110;1;27-37

  • A novel anterograde trafficking signal present in the N-terminal extracellular domain of ionotropic glutamate receptors.

    Xia H, von Zastrow M and Malenka RC

    Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California 94304, USA.

    Trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to and from the postsynaptic membrane plays an important role in regulating transmission at excitatory synapses. AMPA receptor subunits contain a large extracellular N-terminal domain that is important for receptor assembly (). To further investigate the determinants of receptor assembly and surface expression, we have epitope-tagged the N-terminal domain of the AMPA receptor subunit, GluR1, and expressed it in human embryonic kidney 293 cells and hippocampal neurons. Full-length GluR1 was readily detected on the cell surface in both cell types. However, surface expression was profoundly decreased by deletion or replacement of nine amino acids in the extreme N terminus. Immunoprecipitation experiments demonstrated that the mutant GluR1 in which this sequence was deleted still interacts with GluR2, suggesting that mutant GluR1 is capable of at least partial assembly into heteromeric structures. The mutant forms of GluR1 co-localize with an endoplasmic reticulum marker suggesting that they are retained in this structure. These results suggest a specific function of a short sequence present in the N-terminal domain in controlling anterograde trafficking of ionotropic glutamate receptors.

    The Journal of biological chemistry 2002;277;49;47765-9

  • Flip and flop splice variants of AMPA receptor subunits in the spinal cord of amyotrophic lateral sclerosis.

    Tomiyama M, Rodríguez-Puertas R, Cortés R, Pazos A, Palacios JM and Mengod G

    Department of Neurochemistry, Instituto de Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas (IIBB-CSIC, IDIBAPS), 08036 Barcelona, Spain.

    Excitotoxicity mediated by AMPA receptors has been suggested to be implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). To investigate the relevance of AMPA receptors to motor neuron degeneration in ALS, we evaluated the expression of mRNAs coding for flip and flop splice variants of AMPA receptor subunits (GluR-A to GluR-D) in the cervical segment of the spinal cord from control individuals and patients with ALS using in situ hybridization histochemistry. Transcript mRNAs coding for flop variants were significantly decreased in the ventral horn of the spinal cord from patients with ALS, whereas the mRNAs for flip variants were preserved. These findings suggest that the relative abundance of flip variants vs. flop variants is increased in spinal motor neurons of ALS patients when compared to that of control individuals. Flip variants promote assemblies of slowly desensitizing AMPA receptors. These results imply that spinal motor neurons of ALS patients possess more slowly desensitizing AMPA receptors than those of control individuals. This expression change of AMPA receptors in ALS may account for vulnerability of motor neurons in this disease.

    Synapse (New York, N.Y.) 2002;45;4;245-9

  • Blockage of Ca(2+)-permeable AMPA receptors suppresses migration and induces apoptosis in human glioblastoma cells.

    Ishiuchi S, Tsuzuki K, Yoshida Y, Yamada N, Hagimura N, Okado H, Miwa A, Kurihara H, Nakazato Y, Tamura M, Sasaki T and Ozawa S

    Department of Neurosurgery, Gunma University School of Medicine, Maebashi, Gunma, Japan. ishogo@showa.gunma-u.ac.jp

    Glioblastoma multiforme is the most undifferentiated type of brain tumor, and its prognosis is extremely poor. Glioblastoma cells exhibit highly migratory and invasive behavior, which makes surgical intervention unsuccessful. Here, we showed that glioblastoma cells express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors assembled from the GluR1 and/or GluR4 subunits, and that their conversion to Ca(2+)-impermeable receptors by adenovirus-mediated transfer of the GluR2 cDNA inhibited cell locomotion and induced apoptosis. In contrast, overexpression of Ca(2+)-permeable AMPA receptors facilitated migration and proliferation of the tumor cells. These findings indicate that Ca(2+)-permeable AMPA receptors have crucial roles in growth of glioblastoma. Blockage of these Ca(2+)-permeable receptors may be a useful therapeutic strategy for the prevention of glioblastoma invasion.

    Nature medicine 2002;8;9;971-8

  • The PDZ proteins PICK1, GRIP, and syntenin bind multiple glutamate receptor subtypes. Analysis of PDZ binding motifs.

    Hirbec H, Perestenko O, Nishimune A, Meyer G, Nakanishi S, Henley JM and Dev KK

    Department of Anatomy, Medical Research Council Centre of Synaptic Plasticity, Medical School, University of Bristol, Bristol BS8 1TD, United Kingdom.

    Using sequence homology searches, yeast two-hybrid assays and glutathione S-transferase (GST)-pull-down approaches we have identified a series of glutamate receptor subunits that interact differentially with the PDZ proteins GRIP, PICK1, and syntenin. GST-pull-down experiments identified more interactions than detected by yeast two-hybrid assays. We report several receptor-protein interactions, strong ones include: (i) GRIP and syntenin with mGluR7a, mGluR4a, and mGluR6; (ii) PICK1 and GRIP with mGluR3; and (iii) syntenin with all forms of GluR1-4 and mGluR7b. We further characterized the novel mGluR7a-GRIP interaction found both in yeast two-hybrid and GST-pull-down assays and observed that mGluR7a localization overlapped with GRIP with in hippocampal neurons. The wide range of targets for PICK1, GRIP, and syntenin suggests they may represent a molecular mechanism that can concentrate and/or regulate a number of different receptors at a common site on a synapse. These data also suggest that the structural determinants involved in PDZ interactions are more complex than originally envisaged.

    Funded by: Medical Research Council: G9629038

    The Journal of biological chemistry 2002;277;18;15221-4

  • Modeling of the pore domain of the GLUR1 channel: homology with K+ channel and binding of channel blockers.

    Tikhonov DB, Mellor JR, Usherwood PN and Magazanik LG

    Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, 44 Thorez pr., St. Petersburg 194223, Russia. tikhonov@VV3977.spb.edu

    Molecular models of the M2 segments of the GluR1 channel have been elaborated using a molecular mechanics approach. The models are based on the homology between pore-lining segments of AMPA receptor channels and the KcsA K+ channel and on cyclic H bonds at the Q/R site of the AMPA receptor channel. The N-terminal region of an M2 segment of the channel is assumed, like that of the K+ channel, to adopt a helical conformation. Due to a deletion, the C-terminal end of the M2 segment of the AMPA receptor is more stretched than that of the K+ channel. As a result, only a single oxygen ring may be exposed to the AMPA receptor channel pore. Data on the block of AMPA receptor channels by dicationic adamantane derivatives have been used to select the most relevant model. The model with the oxygen of a Gly residue (position +2 from the Q/R site) exposed to the pore best fits the experimental data. This model also fits experimental data for another class of AMPA receptor antagonists, the polyamine amides. According to the model, the side-chains of the C-terminal residues are involved in intra-receptor interactions that stabilize the structure of the channel rather than in interactions with ions in the pore.

    Biophysical journal 2002;82;4;1884-93

  • Nicotine preconditioning antagonizes activity-dependent caspase proteolysis of a glutamate receptor.

    Meyer EL, Gahring LC and Rogers SW

    Salt Lake City Veterans Affairs-Geriatrics Research, Education, and Clinical Center and the University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.

    Neuronal excitation is required for normal brain function including processes of learning and memory, yet if this process becomes dysregulated there is reduced neurotransmission and possibly death through excitotoxicity. Nicotine, through interaction with neuronal nicotinic acetylcholine receptors, possesses the ability to modulate neurotransmitter systems through numerous mechanisms that define this critical balance. We examined the modulatory role of nicotine in primary mixed cortical neuronal-glial cultures on activity-dependent caspase cleavage of a glutamate receptor, GluR1. We find that GluR1, but not GluR2 or GluR3, is a substrate for agonist (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-initiated rapid proteolytic cleavage at aspartic acid 865 through the activation of caspase 8-like activity that is independent of membrane fusion and is not coincident with apoptosis. Dose-dependent nicotine preconditioning for 24 h antagonizes agonist-initiated caspase cleavage of GluR1 through a mechanism that is coincident with desensitization of both nAChRalpha4beta2 and nAChRalpha7 receptors and the delayed activation of a caspase 8-like activity. The modulation of GluR1 agonist-initiated caspase-mediated cleavage by nicotine preconditioning offers a novel insight into how this agent can impart its numerous effects on the nervous system.

    Funded by: NINDS NIH HHS: NS35181

    The Journal of biological chemistry 2002;277;13;10869-75

  • Synapse-associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway.

    Sans N, Racca C, Petralia RS, Wang YX, McCallum J and Wenthold RJ

    Laboratory of Neurochemistry, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892-8027, USA. sansn@nidcd.nih.gov

    The regulation of AMPA receptors at the postsynaptic membrane is a fundamental component of synaptic plasticity. In the hippocampus, the induction of long-term potentiation increases the delivery of GluR1, a major AMPA receptor subunit in hippocampal pyramidal neurons, to the synaptic plasma membrane through a mechanism that requires the PDZ binding domain of GluR1. Synapse-associated protein 97 (SAP97), a member of the membrane-associated guanylate kinase family, is believed to associate with AMPA receptors (AMPARs) containing the GluR1 subunit, but the functional significance of these interactions is unclear. We investigated the interaction of GluR1 with SAP97, the only PDZ protein known to interact with GluR1. We find that interactions involving SAP97 and GluR1 occur early in the secretory pathway, while the receptors are in the endoplasmic reticulum or cis-Golgi. In contrast, few synaptic receptors associate with SAP97, suggesting that SAP97 dissociates from the receptor complex at the plasma membrane. We also show that internalization of GluR1, as triggered by NMDAR activation, does not require SAP97. These results implicate GluR1-SAP97 interactions in mechanisms underlying AMPA receptor targeting.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;19;7506-16

  • Agonist-independent activation of metabotropic glutamate receptors by the intracellular protein Homer.

    Ango F, Prézeau L, Muller T, Tu JC, Xiao B, Worley PF, Pin JP, Bockaert J and Fagni L

    CNRS-UPR 9023, CCIPE, 141 Rue de la Cardonille, 34000 Montpellier, France.

    G-protein-coupled receptors (GPCRs) transduce signals from extracellular transmitters to the inside of the cell by activating G proteins. Mutation and overexpression of these receptors have revealed that they can reach their active state even in the absence of agonist, as a result of a natural shift in the equilibrium between their inactive and active conformations. Such agonist-independent (constitutive) activity has been observed for the glutamate GPCRs (the metabotropic glutamate receptors mGluR1a and mGluR5) when they are overexpressed in heterologous cells. Here we show that in neurons, the constitutive activity of these receptors is controlled by Homer proteins, which bind directly to the receptors' carboxy-terminal intracellular domains. Disruption of this interaction by mutagenesis or antisense strategies, or expression of endogenous Homer1a (H1a), induces constitutive activity in mGluR1a or mGluR5. Our results show that these glutamate GPCRs can be directly activated by intracellular proteins as well as by agonists.

    Nature 2001;411;6840;962-5

  • Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms.

    Chen L, Chetkovich DM, Petralia RS, Sweeney NT, Kawasaki Y, Wenthold RJ, Bredt DS and Nicoll RA

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco 94143, USA.

    Stargazer, an ataxic and epileptic mutant mouse, lacks functional AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate) receptors on cerebellar granule cells. Stargazin, the mutated protein, interacts with both AMPA receptor subunits and synaptic PDZ proteins, such as PSD-95. The interaction of stargazin with AMPA receptor subunits is essential for delivering functional receptors to the surface membrane of granule cells, whereas its binding with PSD-95 and related PDZ proteins through a carboxy-terminal PDZ-binding domain is required for targeting the AMPA receptor to synapses. Expression of a mutant stargazin lacking the PDZ-binding domain in hippocampal pyramidal cells disrupts synaptic AMPA receptors, indicating that stargazin-like mechanisms for targeting AMPA receptors may be widespread in the central nervous system.

    Nature 2000;408;6815;936-43

  • Regulation of AMPA receptor GluR1 subunit surface expression by a 4. 1N-linked actin cytoskeletal association.

    Shen L, Liang F, Walensky LD and Huganir RL

    Howard Hughes Medical Institute, Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    The synaptic localization, clustering, and immobilization of neurotransmitter receptors and ion channels play important roles in synapse formation and synaptic transmission. Although several proteins have been identified that interact with AMPA receptors and that may regulate their synaptic targeting, little is known about the interaction of AMPA receptors with the cytoskeleton. In studies examining the interaction of the AMPA receptor GluR1 subunit with neuronal proteins, we determined that GluR1 interacts with the 4.1G and 4.1N proteins, homologs of the erythrocyte membrane cytoskeletal protein 4.1. Using the yeast two-hybrid system and a heterologous cell system, we demonstrated that both 4.1G and 4.1N bind to a membrane proximal region of the GluR1 C terminus, and that a region within the C-terminal domain of 4.1G or 4.1N is sufficient to mediate the interaction. We also found that 4.1N can associate with GluR1 in vivo and colocalizes with AMPA receptors at excitatory synapses. Disruption of the interaction of GluR1 with 4.1N or disruption of actin filaments decreased the surface expression of GluR1 in heterologous cells. Moreover, disruption of actin filaments in cultured cortical neurons dramatically reduced the level of surface AMPA receptors. These results suggest that protein 4.1N may link AMPA receptors to the actin cytoskeleton.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2000;20;21;7932-40

  • GRASP-1: a neuronal RasGEF associated with the AMPA receptor/GRIP complex.

    Ye B, Liao D, Zhang X, Zhang P, Dong H and Huganir RL

    Howard Hughes Medical Institute, Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, we have identified several GRIP-associated proteins (GRASPs) that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduced the synaptic targeting of AMPA receptors. In addition, the subcellular distribution of both AMPA receptors and GRASP-1 was rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1 may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity.

    Neuron 2000;26;3;603-17

  • Immunolocalization of mGluR1alpha in specific populations of local circuit neurons in the cerebral cortex.

    Stinehelfer S, Vruwink M and Burette A

    Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC 27599, USA.

    By coupling glutamate to the IP(3) signaling pathway, group I metabotropic receptors can increase intracellular Ca(2+) concentration, and might thus contribute to excitotoxicity. To identify neurons that might be vulnerable to such injury, we performed immunofluorescence histochemistry for metabotropic glutamate receptor 1alpha (mGluR1alpha) in the cerebral cortex of adult rat. mGluR1alpha was in somata and dendrites of a subset of non-pyramidal neurons scattered throughout the cerebral cortex. To further characterize mGluR1alpha-positive neurons, we investigated its colocalization with several neurochemical markers. Nearly all mGluR1alpha-positive cells were interneurons immunopositive for gamma-aminobutyric acid. The majority (70-80%) of mGluR1alpha-immunopositive neurons were double-labeled for somatostatin. Approximately half of calretinin-positive neurons and 30% of calbindin-positive neurons expressed mGluR1alpha. In contrast, parvalbumin-expressing neurons were rarely positive for mGluR1alpha. Neurons staining strongly for mGluR1alpha were also positive for GluR1. These results indicated that mGluR1alpha is expressed by specific classes of GABAergic neurons in the neocortex, and suggests a mechanism by which these neurons may be especially vulnerable to excitotoxic injury.

    Funded by: PHS HHS: R01-35527

    Brain research 2000;861;1;37-44

  • Calnexin and the immunoglobulin binding protein (BiP) coimmunoprecipitate with AMPA receptors.

    Rubio ME and Wenthold RJ

    Laboratory of Neurochemistry, NIDCD, NIH, Bethesda, Maryland 20892-4162, USA.

    To identify proteins that interact with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, we carried out coimmunoprecipitation analyses on detergent-solubilized rat forebrain membranes. Membranes were solubilized with Triton X-100, and immunoprecipitation was done using subunit-specific antibodies to GluR1, GluR2/3, and GluR4 attached to protein Aagarose. Proteins bound to the antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining and western blotting. With solubilization in low ionic strength buffer, several coimmunoprecipitating proteins, with Mr = 17,000-100,000, were identified in silver-stained gels. Western blots were then probed with antibodies to a series of candidate proteins that were chosen based on the molecular masses of the copurifying proteins. Two of these were identified as the molecular chaperones calnexin (90 kDa) and the immunoglobulin binding protein (BiP; 78 kDa). Immunoprecipitation with antibodies to calnexin and BiP demonstrated that glycosylated AMPA receptor subunits were associated. The relationship between AMPA receptors and calnexin and BiP was further studied with immunocytochemistry of the hippocampus. Both calnexin and BiP labeling was present not only in the cell body but also in dendrites of hippocampal pyramidal neurons, where double-label immunofluorescence also showed the presence of AMPA receptor subunits.

    Journal of neurochemistry 1999;73;3;942-8

  • Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation.

    Shi SH, Hayashi Y, Petralia RS, Zaman SH, Wenthold RJ, Svoboda K and Malinow R

    Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.

    To monitor changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor distribution in living neurons, the AMPA receptor subunit GluR1 was tagged with green fluorescent protein (GFP). This protein (GluR1-GFP) was functional and was transiently expressed in hippocampal CA1 neurons. In dendrites visualized with two-photon laser scanning microscopy or electron microscopy, most of the GluR1-GFP was intracellular, mimicking endogenous GluR1 distribution. Tetanic synaptic stimulation induced a rapid delivery of tagged receptors into dendritic spines as well as clusters in dendrites. These postsynaptic trafficking events required synaptic N-methyl-D-aspartate (NMDA) receptor activation and may contribute to the enhanced AMPA receptor-mediatedtransmission observed during long-term potentiation and activity-dependent synaptic maturation.

    Science (New York, N.Y.) 1999;284;5421;1811-6

  • Subtype-specific assembly of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits is mediated by their n-terminal domains.

    Leuschner WD and Hoch W

    Max-Planck-Institut für Entwicklungsbiologie, Abteilung Biochemie, Spemannstrasse 35, D-72076 Tübingen, Germany.

    Glutamate receptors (GluR) are oligomeric protein complexes formed by the assembly of four or perhaps five subunits. The rules that govern the selectivity of this process are not well understood. Here, we expressed combinations of subunits from two related GluR subfamilies in COS7 cells, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors. By co-immunoprecipitation experiments, we assessed the ability of AMPA receptor subunits to assemble into multimeric complexes. Subunits GluR1-4 associated with indistinguishable efficiency with each other, whereas the kainate receptor subunits GluR6 and 7 showed a much lower degree of association with GluR1. Using chimeric receptors and truncation fragments of subunits, we show that this assembly specificity is determined by N-terminal regions of these subunits and that the most N-terminal domain of GluR2 together with a membrane anchor efficiently associates with GluR1.

    The Journal of biological chemistry 1999;274;24;16907-16

  • Differential distribution of ionotropic glutamate receptor subunits in the rat olfactory bulb.

    Montague AA and Greer CA

    Department of Neurosurgery, Yale University School of Medicine, New Haven, Connecticut 06520-8082, USA.

    The subcellular localization of ionotropic glutamate receptor (GluR) subunits was examined with light and electron microscopy in the rat olfactory bulb by using antibodies to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunits: GluR1, GluR2/3, and GluR4; and kainate (KA) receptor subunits: GluR5/6/7. Immunoreactivity to GluR1 was heavy in the glomerular layer, moderate in the external plexiform layer, and localized to periglomerular somata and dendrites, short axon somata and dendrites, mitral cell somata, and mitral/tufted dendrites. GluR2/3 immunoreactivity was heavy in the external plexiform and glomerular layers and localized to periglomerular somata and dendrites, mitral cell somata, mitral/tufted dendrites, granule cell somata, and olfactory nerve-associated glia. GluR4 immunoreactivity showed heavy staining in the external plexiform and olfactory nerve layers with localization to mitral cells, mitral/tufted dendritic processes, and olfactory nerve glial processes. GluR5/6/7 immunoreactivity was heavy in the external plexiform layer, moderate in the olfactory nerve and glomerular layers, and localized to granule cells, mitral cells, and mitral/tufted dendritic processes. Ultrastructural immunolabeling for all antibodies examined showed immunoreactivity in the postsynaptic membrane and densities, adjacent dendritic cytoplasm, and somatic cytoplasm. These data demonstrate a highly specific laminar, cellular, and subcellular distribution of ionotropic GluR subunits within the primary afferent and local synaptic circuits of the olfactory bulb. The results are consistent with the notion that the different roles subserved by glutamate in the olfactory bulb are actuated, in part, by a differential distribution of GluR subunits.

    Funded by: NIDCD NIH HHS: DC00210; NIGMS NIH HHS: GM07205; NINDS NIH HHS: NS10174

    The Journal of comparative neurology 1999;405;2;233-46

  • High resolution physical mapping of human HDAC3, a potential tumor suppressor gene in the 5q31 region.

    Mahlknecht U, Bucala R, Hoelzer D and Verdin E

    Laboratory of Medical Biochemistry, The Picower Institute for Medical Research, Manhasset NY, USA. Mahlknecht@em.uni-frankfurt.de

    Histone deacetylases have been described as crucial cofactors of mammalian transcriptional complexes. We have recently identified human histone deacetylase HDAC3 on chromosome 5q31 by fluorescence in situ hybridization (FISH) in a region commonly deleted in malignant myeloid disease. Since HDAC3 carries strong potential to be a tumor suppressor gene, we report herein its exact position between the CD14 and GRIA1 genes within the 5q31.1 subband.

    Cytogenetics and cell genetics 1999;86;3-4;237-9

  • 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

  • SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit.

    Leonard AS, Davare MA, Horne MC, Garner CC and Hell JW

    Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706-1532, USA.

    Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartate-type glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.

    Funded by: NIA NIH HHS: AG12978; NINDS NIH HHS: R01-NS35563

    The Journal of biological chemistry 1998;273;31;19518-24

  • AMPA receptor-mediated excitotoxicity in human NT2-N neurons results from loss of intracellular Ca2+ homeostasis following marked elevation of intracellular Na+.

    Itoh T, Itoh A, Horiuchi K and Pleasure D

    Division of Neurology, The Children's Hospital of Philadelphia, Pennsylvania 19104, USA.

    Human NT2-N neurons express Ca2+-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptors (AMPA-GluRs) and become vulnerable to excitotoxicity when AMPA-GluR desensitization is blocked with cyclothiazide. Although the initial increase in intracellular Ca2+ levels ([Ca2+]i) was 1.9-fold greater in the presence than in the absence of cyclothiazide, Ca2+ entry via AMPA-GluRs in an early phase of the exposure was not necessary to elicit excitotoxicity in these neurons. Rather, subsequent necrosis was caused by a >40-fold rise in [Na+]i, which induced a delayed [Ca2+]i rise. Transfer of the neurons to a 5 mM Na+ medium after AMPA-GluR activation accelerated the delayed [Ca2+]i rise and intensified excitotoxicity. Low-Na+ medium-enhanced excitotoxicity was partially blocked by amiloride or dizocilpine (MK-801), and completely blocked by removal of extracellular Ca2+, suggesting that Ca2+ entry by reverse operation of Na+/Ca2+ exchangers and via NMDA glutamate receptors was responsible for the neuronal death after excessive Na+ loading. Our results serve to emphasize the central role of neuronal Na+ loading in AMPA-GluR-mediated excitotoxicity in human neurons.

    Funded by: NINDS NIH HHS: NS08075, NS25044

    Journal of neurochemistry 1998;71;1;112-24

  • Adjacent asparagines in the NR2-subunit of the NMDA receptor channel control the voltage-dependent block by extracellular Mg2+.

    Wollmuth LP, Kuner T and Sakmann B

    Abteilung Zellphysiologie, Max-Planck-Institut für medizinische Forschung, Heidelberg, Germany. wollmuth@sunny.mpimf-Heidelberg.mpg.de

    1. The voltage-dependent block of N-methyl-D-aspartate (NMDA) receptor channels by extracellular Mg2+ is a critical determinant of its contribution to CNS synaptic physiology. The function of the narrow constriction of the channel in determining the block was investigated by analysing the effects of a set different amino acid substitutions at exposed residues positioned at or near this region. NMDA receptor channels, composed of wild-type and mutant NR1- and NR2A-subunits, were expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. 2. In wild-type channels, the voltage dependence (delta) of the block Mg2+ was concentration dependent with values of delta of integral of 0.82 in 0.07 mM and higher concentrations. Under bionic conditions with high extracellular Mg2+ and K+ as the reference ion, Mg2+ weakly permeated the channel. Over intermediate potentials (approximately -60 to -10 mV), this weak permeability had no apparent effect on the block but at potentials negative to approximately -60mV, it attenuated the extent and voltage dependence of the block. 3. Substitutions of glycine, serine, glutamine or aspartate for the N-site asparagine in the NR1-subunit enhanced the extent of block over intermediate potentials but left the voltage dependence of the block unchanged indicating that structural determinants of the block remained. These same substitutions either attenuated or left unchanged the apparent Mg2+ permeability. 4. In channels containing substitutions of glycine, serine or glutamine for the N-site asparagine in the NR2A-subunit, the block Mg2+ was reduced at negative potentials. Over intermediate potentials, the block was not strongly attenuated except for the glutamine substitution which reduced the voltage dependence of the block to integral of 0.57 in 0.7 mM Mg2+. 5. Equivalent substitutions for the N + 1 site asparagine in the NR2A-subunit strongly attenuated the block over the entire voltage range. In 0.7 mM Mg2+, the voltage dependence of the block was reduced to 0.50 (glycine), 0.53 (serine) and 0.46 (glutamine). 6. Channels containing substitutions of the N-site or N + 1 site asparagines in the NR2A-subunit showed an increased Mg2+ permeability suggesting that these adjacent asparagines form a barrier for inward Mg2+ flux. Changes in this barrier contribute, at least in part, to the mechanism underlying disruption of the block following substitution of these residues. 7. The adjacent NR2A-subunit asparagines are positioned at or near the narrow constriction of the channel. Pore size, however, did not determine how effectively Mg2+ blocks mutant channels. 8. It is concluded that, at the narrow constriction in the NMDA receptor channel, the adjacent NR2A-subunit asparagines, the N-site and N + 1 site, but not the N-site asparagine of the NR1-subunit, form a critical blocking site for extracellular Mg2+. The contribution to the blocking site, in contrast to the prevailing view, is stronger for the N + 1 site than for the N-site asparagine. The block may involve binding of Mg2+ to these residues.

    The Journal of physiology 1998;506 ( Pt 1);13-32

  • Expression and heteromeric interactions of non-N-methyl-D-aspartate glutamate receptor subunits in the developing and adult cerebellum.

    Ripellino JA, Neve RL and Howe JR

    Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8066, USA.

    The localization and expression of ionotropic non-N-methyl-D-aspartate glutamate receptors (GluR) were investigated in the developing and adult rat cerebellum using subunit-specific polyclonal antibodies for immunocytochemical, immunoblot and immunoprecipitation studies. In P7 animals, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor immunoreactivity was detected in all layers of the cerebellar cortex with the exception of the external granule cell layer. Antibodies against the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunits GluR1 and GluR4 gave strong immunoreactive staining of Bergmann glia in both young and adult animals, and both antibodies showed prominent staining of the molecular layer in the adult cerebellum. Dense immunoreactive staining of Purkinje cell somata and dendrites was obtained with anti-GluR2/3/4c in both the developing and adult cerebellum. Whereas each of the three alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antibodies stained the internal, but not the external, granule cell layer, immunostaining for the kainate-type subunits GluR6/7 and KA2 was detected in both the external and internal granule cell layers. as well as in the molecular layer in both P7 and adult cerebellum. Immunoblot analysis of total cerebellar protein indicated that the level of GluR4 expression increased 15-fold from P1 to P18, whereas the expression of the KA2 subunit protein was nine-fold lower in adult cerebellum than it was at P1. The expression of GluR1 increased moderately (two-fold) from P1 to adult. Subunit interactions between GluR1 and GluR4, as well as between GluR6/7 and KA2, were demonstrated in immunoprecipitation experiments; and the GluR4 and KA2 subunits appear to be present exclusively in heteromeric assemblies with GluR1 and GluR6/7, respectively. The results show that the various alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- and kainate-type subunits are differentially expressed during cerebellar development and further define the possible subunit composition of non-N-methyl-D-aspartate receptors in the major cerebellar cell types.

    Funded by: NICHD NIH HHS: HD24236; NINDS NIH HHS: NS12327, NS30996

    Neuroscience 1998;82;2;485-97

  • Identification of the Ca2+/calmodulin-dependent protein kinase II regulatory phosphorylation site in the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-type glutamate receptor.

    Barria A, Derkach V and Soderling T

    Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, USA.

    Ca2+/CaM-dependent protein kinase II (CaM-KII) can phosphorylate and potentiate responses of alpha-amino3-hydroxyl-5-methyl-4-isoxazole-propionate-type glutamate receptors in a number of systems, and recent studies implicate this mechanism in long term potentiation, a cellular model of learning and memory. In this study we have identified this CaM-KII regulatory site using deletion and site-specific mutants of glutamate receptor 1 (GluR1). Only mutations affecting Ser831 altered the 32P peptide maps of GluR1 from HEK-293 cells co-expressing an activated CaM-KII. Likewise, when CaM-KII was infused into cells expressing GluR1, the Ser831 to Ala mutant failed to show potentiation of the GluR1 current. The Ser831 site is specific to GluR1, and CaM-KII did not phosphorylate or potentiate current in cells expressing GluR2, emphasizing the importance of the GluR1 subunit in this regulatory mechanism. Because Ser831 has previously been identified as a protein kinase C phosphorylation site (Roche, K. W., O'Brien, R. J., Mammen, A. L., Bernhardt, J., and Huganir, R. L. (1996) Neuron 16, 1179-1188), this raises the possibility of synergistic interactions between CaM-KII and protein kinase C in regulating synaptic plasticity.

    Funded by: NINDS NIH HHS: NS 27037

    The Journal of biological chemistry 1997;272;52;32727-30

  • 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

  • Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit.

    Roche KW, O'Brien RJ, Mammen AL, Bernhardt J and Huganir RL

    Department of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    We have characterized the phosphorylation of the glutamate receptor subunit GluR1, using biochemical and electrophysiological techniques. GluR1 is phosphorylated on multiple sites that are all located on the C-terminus of the protein. Cyclic AMP-dependent protein kinase specifically phosphorylates SER-845 of GluR1 in transfected HEK cells and in neurons in culture. Phosphorylation of this residue results in a 40% potentiation of the peak current through GluR1 homomeric channels. In addition, protein kinase C specifically phosphorylates Ser-831 of GluR1 in HEK-293 cells and in cultured neurons. These results are consistent with the recently proposed transmembrane topology models of glutamate receptors, in which the C-terminus is intracellular. In addition, the modulation of GluR1 by PKA phosphorylation of Ser-845 suggests that phosphorylation of this residue may underlie the PKA-induced potentiation of AMPA receptors in neurons.

    Funded by: NINDS NIH HHS: 1T32NS07368

    Neuron 1996;16;6;1179-88

  • Identification of a Ca2+/calmodulin-dependent protein kinase II regulatory phosphorylation site in non-N-methyl-D-aspartate glutamate receptors.

    Yakel JL, Vissavajjhala P, Derkach VA, Brickey DA and Soderling TR

    Vollum Institute, Oregon Health Sciences University, Portland 97201.

    Glutamate receptor ion channels are colocalized in postsynaptic densities with Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II), which can phosphorylate and strongly enhance non-N-methyl-D-aspartate (NMDA) glutamate receptor current. In this study, CaM-kinase II enhanced kainate currents of expressed glutamate receptor 6 in 293 cells and of wild-type glutamate receptor 1, but not the Ser-627 to Ala mutant, in Xenopus oocytes. A synthetic peptide corresponding to residues 620-638 in GluR1 was phosphorylated in vitro by CaM-kinase II but not by cAMP-dependent protein kinase or protein kinase C. The 32P-labeled peptide map of this synthetic peptide appears to be the same as the two-dimensional peptide map of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptors phosphorylated in cultured hippocampal neurons by CaM-kinase II described elsewhere. This CaM-kinase II regulatory phosphorylation site is conserved in all AMPA/kainate-type glutamate receptors, and its phosphorylation may be important in enhancing postsynaptic responsiveness as occurs during synaptic plasticity.

    Funded by: NIDDK NIH HHS: DK07680; NINDS NIH HHS: NS27037

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;5;1376-80

  • AMPA glutamate receptors and their flip and flop mRNAs in human hippocampus.

    Eastwood SL, Burnet PW, Beckwith J, Kerwin RW and Harrison PJ

    University Department of Psychiatry, Warneford Hospital, Oxford, UK.

    AMPA-preferring glutamate receptor genes gluR1-4 undergo alternative splicing into flip and flop mRNA isoforms. In the rodent hippocampus, gluR1 and gluR2 isoforms are differentially expressed. We have studied their distribution in human hippocampus. As in the rat, flop isoforms predominate over flip in dentate gyrus, whereas gluR1 flip is prominent only in CA3. In contrast to the rat, flop mRNAs are clearly present in CA3. At a cellular level, pyramidal neurones express moderate amounts of each isoform. In several hippocampal fields, scattered non-pyramidal cells--putatively interneurones and glia--show abundant expression. The findings are supported by immunocytochemical detection of gluR1 and gluR2/3. As the four encoded isoforms have distinct properties, their differential expression within the hippocampus, and between species, should be taken into account when considering the roles of AMPA receptors in normal and abnormal brain states.

    Funded by: Wellcome Trust

    Neuroreport 1994;5;11;1325-8

  • Transmembrane topology of the glutamate receptor subunit GluR6.

    Roche KW, Raymond LA, Blackstone C and Huganir RL

    Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

    Ionotropic glutamate receptors mediate most rapid excitatory synaptic transmission in the mammalian central nervous system. These receptors are divided into alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-D-aspartate receptors based on pharmacological and electrophysiological characteristics. Ionotropic receptor subunits are integral membrane proteins that have been proposed to have a large extracellular ligand-binding N-terminal domain, four hydrophobic transmembrane domains, and an extracellular C-terminal domain. In this study we have shown that both AMPA receptor subunits (GluR1-4) and kainate receptor subunits (GluR6/7) are glycosylated in adult rat brain; however, the kainate receptor subunits are glycosylated to a greater extent. Examination of the sequences of AMPA and kainate receptors revealed that kainate receptors have several additional consensus sites for N-linked glycosylation; interestingly, one of these is located in the proposed major intracellular loop of the receptor subunits. To test the proposed transmembrane topology model for these receptors, we have used site-specific mutagenesis of the GluR6 subunit to remove the consensus glycosylation site located within the proposed intracellular loop. Mutagenesis of this site demonstrates that it is glycosylated in transiently transfected human embryonic kidney cells, which express functional kainate receptors. Since N-linked glycosylation has only been found to occur on extracellular domains of plasma membrane proteins, these results suggest that the proposed transmembrane topology model for the glutamate receptor subunits is incorrect. Combining these results with other recent data, we have proposed an alternative transmembrane topology model.

    Funded by: NIGMS NIH HHS: GM-07309

    The Journal of biological chemistry 1994;269;16;11679-82

  • Biochemical and assembly properties of GluR6 and KA2, two members of the kainate receptor family, determined with subunit-specific antibodies.

    Wenthold RJ, Trumpy VA, Zhu WS and Petralia RS

    Laboratory of Neurochemistry, National Institute of Deafness and Communication Disease, National Institutes of Health, Bethesda, Maryland 20892.

    To examine subunit assembly and biochemical properties of two members of the kainate family of glutamate receptors (GluR), antibodies were made to synthetic peptides corresponding to the carboxyl termini of GluR6 and KA2. Immunoblot analysis of membranes from human embryonic kidney cells transfected with glutamate receptor cDNAs showed that these antibodies are selective for their respective receptor subunit except that the antibody to GluR6 also recognizes GluR7, which is expected due to the sequence homology between the two subunits at the carboxyl terminus. In transfected cell membranes, immunoblot analysis with the antibody to GluR6 showed a major immunoreactive band at 118 kDa and minor bands at 103 and 28 kDa. The 103-kDa band appears to be a deglycosylated form of GluR6 since deglycosylation eliminates staining at 118 kDa and increases staining of the 103-kDa band. Immunoblot analysis of KA2 transfected cell membranes shows a major band at 123 kDa and minor bands at 109 and 37 kDa. Deglycosylation converts the 123-kDa band into a 109-kDa band. Analysis of brain tissues shows that both antibodies label single major bands which migrate at the same molecular masses as those from transfected cell membranes, 118 and 123 kDa for GluR6 and KA2, respectively. Immunoprecipitation studies showed that antibodies to GluR6 and KA2 selectively immunoprecipitated [3H]kainate binding activity, but not 3H-labeled alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) binding activity, from Triton X-100-solubilized rat brain membranes. Furthermore, each antibody coimmunoprecipitated GluR6 and KA2 from cells co-transfected with GluR6 and KA2 cDNAs and from detergent-solubilized rat brain membranes, indicating that these two subunits can coassemble into a molecular complex. Interestingly, GluR1 and GluR2, subunits of the AMPA receptor, also co-immunoprecipitated with GluR6 in cells co-transfected with GluR6 and GluR1 or GluR2 cD-NAs. Such complexes appear to be present to a limited extent in the brain.

    The Journal of biological chemistry 1994;269;2;1332-9

  • Expression of alternatively-spliced glutamate receptors in human hippocampus.

    McLaughlin DP, Cheetham ME and Kerwin RW

    Department of Neuroscience, Institute of Psychiatry, London, UK.

    Rat glutamate receptors have been shown to be expressed as two developmentally regulated, alternatively spliced isoforms. We have investigated the expression of these isoforms of GluRA and GluRB in the human hippocampus. The expression pattern of the mRNAs coding for these subunits does not correspond to that in the rat hippocampus, both isoforms being preferentially expressed in the dentate gyrus and CA1 regions, with lower expression in CA3, with the exception of GluRB flop, where hybridization in CA3 is only lower than in dentate gyrus. Cloning of cDNA from human frontal cortex has also revealed that the two isoforms of human GluRB have virtual nucleotide sequence identity with the alternative exons in the rat, confirming the usefulness of oligonucleotides complementary to the rat cDNAs as probes for these receptor subunits in human neuropsychiatric disorders.

    European journal of pharmacology 1993;244;1;89-92

  • Chromosomal localization of human glutamate receptor genes.

    McNamara JO, Eubanks JH, McPherson JD, Wasmuth JJ, Evans GA and Heinemann SF

    Department of Veterans Affairs Medical Center, Durham, NC.

    The chromosomal localization of human glutamate receptor genes (GluR1-4) has been established using PCR with DNA isolated from mapping panels of Chinese hamster-human hybrid cell lines and high-resolution fluorescent in situ suppression hybridization. This was accomplished with genomic clones containing putative human homologs of rat GluR 1-4 isolated by high-stringency screening of a cosmid library with the rat cDNAs encoding GluR1-4. The locations of GluR1-4, respectively, are 5q32-33, 4q32-33, Xq25-26, and 11q22-23. Evidence implicating glutamatergic synapses in a diversity of physiologic and pathologic processes together with concordance of the chromosomal locales and results of linkage analyses establishes GluR3 and GluR4 as candidate genes for a number of nervous system disorders including the oculocerebral-renal syndrome of Lowe and a form of manic-depressive illness.

    Funded by: NHGRI NIH HHS: HG00320; NINDS NIH HHS: NS17771, NS24448; ...

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1992;12;7;2555-62

  • Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors.

    Sun W, Ferrer-Montiel AV, Schinder AF, McPherson JP, Evans GA and Montal M

    Department of Biology, University of California, San Diego, La Jolla 92093.

    A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCR analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists with the following specificity sequence: domoate greater than kainate much greater than quisqualate greater than or equal to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid greater than or equal to L-glutamate much greater than N-methyl-D-aspartate. The kainate-elicited currents were specifically blocked by 6-cyano-7-nitroquinoxaline-2,3-dione but were insensitive to 2-amino-5-phosphonovalerate and kynurenic acid. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.

    Funded by: NHGRI NIH HHS: HG00047; NIGMS NIH HHS: GM33868; NIMH NIH HHS: MH-44638

    Proceedings of the National Academy of Sciences of the United States of America 1992;89;4;1443-7

  • The human glutamate receptor cDNA GluR1: cloning, sequencing, expression and localization to chromosome 5.

    Potier MC, Spillantini MG and Carter NP

    Medical Research Council, Laboratory of Molecular Biology, Cambridge, England.

    The rat glutamate receptor is a 907 amino acid transmembrane protein. Using the rat GluR1 cDNA as a probe, we have isolated cDNA clones from a human hippocampal cDNA library. Sequence of a full length cDNA clone revealed 98.2% and 89.4% identity to the rat sequence at the amino acid and nucleotide levels respectively. The human cDNA clone detected an RNA transcript in human cerebral cortex, hippocampus and cerebellum, similar to that seen in rat. In situ hybridization experiments showed that human GluR1 mRNA is present in granule and pyramidal cells in the hippocampal formation and that there is no apparent difference of distribution between control patient and patient with Alzheimer's disease. Dot blot analysis of flow-sorted human chromosomes showed that the GluR1 gene maps to chromosome 5.

    DNA sequence : the journal of DNA sequencing and mapping 1992;2;4;211-8

  • Molecular cloning and chromosomal localization of one of the human glutamate receptor genes.

    Puckett C, Gomez CM, Korenberg JR, Tung H, Meier TJ, Chen XN and Hood L

    Division of Biology, California Institute of Technology, Pasadena 91125.

    Glutamate receptors are the predominant excitatory neurotransmitter receptors in the mammalian brain and are classified on the basis of their activation by different agonists. The agonists kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid define a class of glutamate receptors termed kainate receptors. We have isolated and sequenced a human glutamate receptor (GluHI) cDNA and determined the chromosomal localization of its gene. The DNA sequence of GluHI would encode a 907-amino acid protein that has a 97% identity to one of the rodent kainate receptor subunits. Many of the changes between the predicted amino acid sequence of GluHI and the most similar rodent kainate receptor (GluRI) occur in a region of the protein encoded in rodents by an alternatively spliced exon. The extreme conservation between the human and rat kainate receptor subunits suggests that a similar gene family will encode human kainate receptors. The GluHI mRNA is widely expressed in human brain. The human gene encoding the GluHI subunit is located at 5q33. While the GluHI gene is not located near a chromosomal region associated with any human neurogenetic disorders, the homologous region on mouse chromosome 11 contains the sites of five neurologic mutations.

    Funded by: NIA NIH HHS: S-P50-AGO5142; NINDS NIH HHS: NS 01163-44; PHS HHS: R29 H600037-01

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;17;7557-61

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
L00000014 G2C Homo sapiens Human ARC Human orthologues of mouse AMPA receptor complex adapted from Collins et al (2006) 9
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
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
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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