Enrichment of N-methyl-d-aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities

Al‐Hallaq, Rana A.; Yasuda, Robert P.; Wolfe, Barry B.

doi:10.1046/j.1471-4159.2001.t01-1-00210.x

Cited by 17 publications

(10 citation statements)

References 60 publications

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Section: Discussionsupporting

confidence: 88%

“…This possibility, suggested by the demonstrated heterogeneity of NMDARs (Sheng, et al, 1994) is supported by evidence that ethanol modulates the surface expression of NR1C2‘ and NR2A (Honse et al, 2003), a subunit combination that is also suggested by our results (Figures 3 and 4), although other biochemical data indicates no preferential NR1/NR2 co-assembly (Blahos and Wenthold, 1996; Al-Hallaq et al, 2001). This discrepancy was attributed to different model systems used in their experiments (Al-Hallaq et al, 2001). Light microscopic studies in rat and primate retina suggested a slight preference of NR2A for NR1C2‘ over NR1C2 (Fletcher et al, 2000; Gründer et al, 2000), again consistent with the present results, although immunopositive dendrites were not identified in those studies and so could have originated from either RGCs or amacrine cells.…”

Section: Discussionsupporting

confidence: 88%

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Subunit- and Pathway-Specific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina

Zhang¹,

Diamond²

2009

J. Neurosci.

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Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from ON and OFF bipolar cells in distinct sublaminae of the inner plexiform layer (IPL). AMPA and NMDA receptors (AMPARs and NMDARs) mediate excitatory inputs in both synaptic layers, but specific roles for NMDARs at RGC synapses remain unclear. NMDARs comprise NR1 and NR2 subunits and are anchored by membrane associated guanylate kinases (MAGUKs), but it is unknown whether particular NR2 subunits associate preferentially with particular NR1 splice variants and MAGUKs. Here, we used postembedding immunogold electron microscopy (EM) techniques to examine the subsynaptic localization of NMDAR subunits and MAGUKs at ON and OFF synapses onto rat RGCs. We found that the NR2A subunit, the NR1C2‘ splice variant and MAGUKs PSD-95 and PSD-93 are localized to the postsynaptic density (PSD), preferentially at OFF synapses, whereas the NR2B subunit, the NR1C2 splice variant and the MAGUK SAP102 are localized perisynaptically, with NR2B exhibiting a preference for ON synapses. Consistent with these anatomical data, spontaneous EPSCs (sEPSCs) recorded from OFF cells exhibited an NMDAR component that was insensitive to the NR2B antagonist Ro 25-6981. In ON cells, sEPSCs expressed an NMDAR component, partially sensitive to Ro 25-6981, only when glutamate transport was inhibited, indicating perisynaptic expression of NR2B NMDARs. These results provide the first evidence for preferential association of particular NR1 splice variants, NR2 subunits and MAGUKs at central synapses and suggest that different NMDAR subtypes may play specific roles at functionally distinct synapses in the retinal circuitry.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionsupporting

confidence: 88%

Subunit- and Pathway-Specific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina

Zhang¹,

Diamond²

2009

J. Neurosci.

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“…Relatively higher uses of the C2Ј cassette in the cerebellum and of the C1 and C2 cassettes in the cerebral cortex are generally consistent with previous studies by in situ hybridization and immunoblot analyses (Laurie and Seeburg, 1994;Al-Hallaq et al, 2001). In the present study, C-terminal cassette-dependent differences of the GluR1 subunit were also appreciated in the brain in vivo.…”

Section: Terminus-dependent Self-er Export Mechanisms In Neurons Insupporting

confidence: 92%

NMDA Receptor GluRϵ/NR2 Subunits Are Essential for Postsynaptic Localization and Protein Stability of GluRζ1/NR1 Subunit

Abe¹,

Fukaya²,

Yagi³

et al. 2004

J. Neurosci.

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In NMDA receptors, GluR⑀/NR2 subunits strictly require the GluR1/NR1 subunit to exit from endoplasmic reticulum (ER) to the cell surface in vitro and to the postsynapse in vivo, whereas C terminus-dependent self-surface delivery has been demonstrated for the GluR1 subunit in vitro. To test whether this leads to C terminus-dependent self-postsynaptic expression in neurons in vivo, we investigated the GluR1 subunit in cerebellar granule cells lacking two major GluR⑀ subunits, GluR⑀1/NR2A and GluR⑀3/NR2C. In the mutant cerebellum, synaptic labeling for the GluR1 subunit containing the C2 (GluR1-C2) or C2Ј (GluR1-C2Ј) cassette was reduced at mossy fiber-granule cell synapses to the extrasynaptic level. The loss was not accompanied by decreased transcription and translation levels, increased extrasynaptic labeling, or ER accumulation. Quantitative immunoblot revealed substantial reductions in the mutant cerebellum of GluR1-C2 and GluR1-C2Ј. The most severe deficit was observed in the postsynaptic density (PSD) fraction: mutant levels relative to the wild-type level were 12.3 Ϯ 3.3% for GluR1-C2 and 17.0 Ϯ 4.6% for GluR1-C2Ј. The GluR1 subunit carrying the C1 cassette (GluR1-C1) was, although low in cerebellar content, also reduced to 12.7 Ϯ 3.5% in the mutant PSD fraction. Considering a trace amount of other GluR⑀ subunits in the mutant cerebellum, the severe reductions thus represent that the GluR1 subunit, by itself, is virtually unable to accumulate at postsynaptic sites, regardless of C-terminal forms. By protein turnover analysis, the degradation of the GluR1 subunit was accelerated in the mutant cerebellum, being particularly rapid for that carrying the C2 cassette. Therefore, accompanying expression of GluR⑀ subunits is essential for postsynaptic localization and protein stability of the GluR1 subunit.

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“…Dishes (100 mm) of HEK293 cells (Invitrogen) were transfected and harvested as described previously (Al-Hallaq et al, 2001). Protein concentrations were determined using the bicinchoninic acid method (Pierce).…”

Section: Methodsmentioning

confidence: 99%

“…Immunoblotting was carried out as described previously (Al-Hallaq et al, 2001) with the following modifications. Standard curves using membranes prepared either from HEK293 cells transfected with NR3A cDNA or adult rat cortex were loaded at 1.6-fold dilutions.…”

Section: Methodsmentioning

confidence: 99%

Association of NR3A with theN-Methyl-d-aspartate Receptor NR1 and NR2 Subunits

Al‐Hallaq

Jarabek

et al. 2002

Mol Pharmacol

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The NR3A subunit of the N-methyl-D-aspartate receptor has been shown to form glutamatergic receptor complexes with NR1 and NR2 subunits and excitatory glycinergic receptor complexes with NR1 alone. We developed an antibody to NR3A and, using quantitative immunoblotting techniques, determined the degree of association between the NR3A subunit and the NR1 and NR2 subunits as well as changes in these associations during development. NR3A expression peaks between postnatal days 7 and 10 in the cortex, midbrain, and hippocampus and reaches higher maximal expression levels in these areas than in the olfactory bulb and cerebellum. Immunoprecipitation experiments with an anti-NR1 antibody demonstrated that the majority of NR3A is associated with NR1 in postnatal day 10 rat cortex (80 Ϯ 8%), decreasing by half (38 Ϯ 4%) in the adult rat cortex. Using the anti-NR3A antibody in immunoprecipitation studies, we find that 9.7 Ϯ 0.8% of NR1, 8.7 Ϯ 1.8% of NR2A, and 5.0 Ϯ 0.6% of NR2B are associated with NR3A at postnatal day 10. These values decrease by about half in adult rat cortex. The results of this study demonstrate that NR3A is expressed, distributed, and associated with other subunits in a manner that supports its role in synaptic transmission throughout the rat brain, perhaps playing different roles during development.The glutamate receptor family mediates the majority of fast excitatory synaptic transmission in the mammalian central nervous system. The ionotropic glutamate receptors are divided pharmacologically into three major groups: the Nmethyl-D-aspartate (NMDA) receptor (NMDAR), the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, and the kainate receptor. The NMDAR has received much attention because of its involvement in neuronal development, a variety of neurodegenerative diseases, and certain types of excitotoxicity (Dingledine et al., 1999;Cull-Candy et al., 2001). A voltage-dependent magnesium block and relatively high calcium permeability have implicated the NMDAR in a mechanism thought to be critical for certain types of learning and memory, namely long-term potentiation (McBain and Mayer, 1994).The NMDAR complex is a tetrameric or pentameric structure composed of at least two NR1 subunits and two or three subunits from the NR2 family (NR2A-D) (for review, see McBain and Mayer, 1994;Dingledine et al., 1999). The NR1 subunit is expressed throughout the central nervous system and is required for the formation of functional receptors.

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Enrichment of N-methyl-d-aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities

Cited by 17 publications

References 60 publications

Subunit- and Pathway-Specific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina

Subunit- and Pathway-Specific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina

NMDA Receptor GluRϵ/NR2 Subunits Are Essential for Postsynaptic Localization and Protein Stability of GluRζ1/NR1 Subunit

Association of NR3A with theN-Methyl-d-aspartate Receptor NR1 and NR2 Subunits

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