AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

NR2A-containing NMDA receptors depress glutamatergic synaptic transmission and evoked-dopamine release in the mouse striatum

NMDA receptors play essential roles in the physiology and pathophysiology of the striatum, a brain nucleus involved in motor control and reward-motivated behaviors. NMDA receptors are composed of NR1 and NR2A–D subunits. Functional properties of NMDA receptors are determined by the type of NR2 subunit they contain. In this study, we have examined the involvement of NR2B and NR2A in the modulatory effect of NMDA on glutamatergic and dopaminergic synaptic transmission in the striatum. We found that bath application of NMDA decreased the amplitude of the field excitatory post-synaptic potential/population spike (fEPSP/PS) measured in corticostriatal mouse brain slices. This depression was not affected by the NR2B-selective antagonists Ifenprodil and Ro 25-6981, but was abolished by the NR2A antagonist NVP-AAM077. Activation of corticostriatal neurons by NMDA did not contribute to synaptic depression because similar results were obtained in decorticated striatal slices. Synaptic depression was not dependent on GABA release because the GABA_A receptor antagonist bicuculline did not affect NMDA-induced decrease of the fEPSP/PS. NMDA also depressed evoked-dopamine release through NR2A- but not NR2B-containing NMDA receptors. Our results identify an important role for NR2A-containing NMDA receptors intrinsic to the striatum in regulating glutamatergic synaptic transmission and evoked-dopamine release.     

Tumor necrosis factor-α-induced neutral sphingomyelinase-2 modulates synaptic plasticity by controlling the membrane insertion of NMDA receptors

The insertion and removal of NMDA receptors from the synapse are critical events that modulate synaptic plasticity. While a great deal of progress has been made on understanding the mechanisms that modulate trafficking of NMDA receptors, we do not currently understand the molecular events required for the fusion of receptor containing vesicles with the plasma membrane. Here, we show that sphingomyelin phosphodiesterase 3 (also known as neutral sphingomyelinase-2) is critical for tumor necrosis factor (TNF) α-induced trafficking of NMDA receptors and synaptic plasticity. TNFα initiated a rapid increase in ceramide that was associated with increased surface localization of NMDA receptor NR1 subunits and a specific clustering of NR1 phosphorylated on serines 896 and 897 into lipid rafts. Brief applications of TNFα increased the rate and amplitude of NMDA-evoked calcium bursts and enhanced excitatory post-synaptic currents. Pharmacological inhibition or genetic mutation of neutral sphingomyelinase-2 prevented TNFα-induced generation of ceramide, phosphorylation of NR1 subunits, clustering of NR1, enhancement of NMDA-evoked calcium flux and excitatory post-synaptic currents.     

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.     

Differential regulation of the NMDA receptor by acute and sub-chronic phencyclidine administration in the developing rat

Neurodegeneration induced by the NMDA receptor antagonist, phencyclidine (PCP), has been used to model the pathogenesis of schizophrenia in the developing rat. Acute and sub-chronic administration of PCP in perinatal rats results in different patterns of neurodegeneration. The potential role of an alteration in the membrane expression of NMDA receptors in PCP-induced degeneration is unknown. Acute PCP treatment on postnatal day 7 increased membrane levels of both NMDA receptor subunit 1 (NR1) and NMDA receptor subunit 2B (NR2B) proteins in the frontal cortex; conversely, NR1 and NR2B protein levels in the endoplasmic reticulum fraction were decreased. Acute PCP administration also resulted in increased membrane cortical protein levels of post-synaptic density-95, as well as the activation of calpain, which paralleled the observed increase in membrane expression of NR1 and NR2B. Further, administration of the calpain inhibitor, MDL28170, prevented PCP-induced up-regulation of NR1 and NR2B. On the other hand, sub-chronic PCP treatment on postnatal days 7, 9 and 11 caused an increase in NR1 and NR2A expression, which was accompanied by an increase in both NR1 and NR2A in the endoplasmic reticulum fraction. Sub-chronic PCP administration did not alter levels of post-synaptic density-95 and had no effect on activation of calpain. These data suggest that increased trafficking accounts for up-regulation of cortical NR1/NR2B subunits following acute PCP administration, while increased protein synthesis likely accounts for the increased expression of NR1/NR2A following sub-chronic PCP treatment of the developing rat. These results are discussed in the context of the differential neurodegeneration caused by acute and subchronic PCP administration in the developing rat brain.     

Regulation of dopamine D1 receptor trafficking and desensitization by oligomerization with glutamate N-methyl-D-aspartate receptors

Activation of dopamine D1 receptors is critical for the generation of glutamate-induced long-term potentiation at corticostriatal synapses. In this study, we report that, in striatal neurons, D1 receptors are co-localized with N-methyl-d-aspartate (NMDA) receptors in the postsynaptic density and that they co-immunoprecipitate with NMDA receptor subunits from postsynaptic density preparations. Using modified bioluminescence resonance energy transfer, we demonstrate that D1 and NMDA receptor clustering reflects the existence of direct interactions. The tagged D1 receptor and NR1 subunit cotransfected in COS-7 cells generated a significant bioluminescence resonance energy transfer signal that was insensitive to agonist stimulation and that did not change in the presence of the NR2B subunit, suggesting that the D1 receptor constitutively and selectively interacts with the NR1 subunit of the NMDA channel. Oligomerization with the NR1 subunit substantially modified D1 receptor trafficking. In individually transfected HEK293 cells, NR1 was localized in the endoplasmic reticulum, whereas the D1 receptor was targeted to the plasma membrane. In cotransfected cells, both the D1 receptor and NR1 subunit were retained in cytoplasmic compartments. In the presence of the NR2B subunit, the NR1-D1 receptor complex was translocated to the plasma membrane. These data suggest that D1 and NMDA receptors are assembled within intracellular compartments as constitutive heteromeric complexes that are delivered to functional sites. Coexpression with NR1 and NR2B subunits also abolished agonist-induced D1 receptor cytoplasmic sequestration, indicating that oligomerization with the NMDA receptor could represent a novel regulatory mechanism modulating D1 receptor desensitization and cellular trafficking.     

Spatial memory formation induces recruitment of NMDA receptor and PSD-95 to synaptic lipid rafts

NMDA receptors (NMDARs) activation in the hippocampus and insular cortex is necessary for spatial memory formation. Recent studies suggest that localization of NMDARs to lipid rafts enhance their signalization, since the kinases that phosphorylate its subunits are present in larger proportion in lipid raft membrane microdomains. We sought to determine the possibility that NMDAR translocation to synaptic lipid rafts occurs during plasticity processes such as memory formation. Our results show that water maze training induces a rapid recruitment of NMDAR subunits (NR1, NR2A, NR2B) and PSD-95 to synaptic lipid rafts and decrease in the post-synaptic density plus an increase of NR2B phosphorylation at tyrosine 1472 in the rat insular cortex. In the hippocampus, spatial training induces selective translocation of NR1 and NR2A subunits to lipid rafts. These results suggest that NMDARs translocate from the soluble fraction of post-synaptic membrane (non-raft PSD) to synaptic lipid raft during spatial memory formation. The recruitment of NMDA receptors and other proteins to lipid rafts could be an important mechanism for increasing the efficiency of synaptic transmission during synaptic plasticity process.     

受体、突触和记忆

大脑中神经元突触间的信号传递是由许多神经递质受体介导的。在过去,Richard L．Huganir实验室一直致力于神经递质受体功能调节的分子机制。而最近,该实验室又聚焦到大脑中一种最主要的兴奋性受体的研究——谷氨酸受体。谷氨酸受体主要可以分为两大类：AMPA受体和NMDA受体。AMPA受体主要介导了快速的兴奋性突触传递;而NMDA受体则在神经可塑性和发育中起到重要作用。实验发现,AMPA受体和NMDA受体都可以被一系列的蛋白激酶磷酸化,而磷酸化的水平则直接影响了这些受体的功能特性,包括通道电导和受体膜定位等。AMPA受体磷酸化的水平同时还在学习和记忆的细胞模型中发生改变,如长时程增强（LTP）和长时程抑制（LTD）。此外,AMPA受体中GluR1亚单位的磷酸化对于各种形式的可塑性以及空间记忆的维持有重要的作用。实验室主要研究突触部位谷氨酸受体在亚细胞水平的定位和聚集的分子机制。最近,一系列可以直接或间接与AMPA和NMDA受体相互作用的蛋白质得以发现,其中包括一个新发现的蛋白家族GRIPs（glutamate receptor interacting proteins）。GRIPs可以直接和AMPA受体的GluR2／3亚单位的C端结合。GRIPs包含7个PDZ结构域,可以介导蛋白与蛋白直接的相互连接,从而把各个AMPA受体交互连接在一起并与其他蛋白相连。另外,GluR2亚单位的c端还可以和兴奋性突触中的蛋白激酶C结合蛋白（PICK1）的PDZ结构域相互作用。另外,GluR2亚单位的C端也可以与一种参与膜融合的蛋白NSF相互作用。这些与AMPA受体相互作用的蛋白质对于受体在膜上的运输以及定位有至关重要的作用。同时,受体与PICK1和GRIP的结合对于小脑运动学习中的LTD有重要作用。总体上说,该实验室发现了一系列可以调节神经递质受体功能的分子机制,这些工作提示受体功能的调节可能是?     

Identification of an N-Methyl-d-aspartate Receptor in Isolated Nervous System Mitochondria

NMDA ionotropic glutamate receptors gate the cytoplasmic influx of calcium, which may, depending on the intensity of the stimulus, subserve either normal synaptic communication or cell death. We demonstrate that when isolated mitochondria are exposed to calcium and NMDA agonists, there is a significant increase in mitochondrial calcium levels. The agonist/antagonist response studies on purified mitochondria suggest the presence of a receptor on mitochondria with features similar to plasma membrane NMDA receptors. Immunogold electron microscopy of hippocampal tissue sections revealed extensive localization of NR2a subunit immunoreactivity on mitochondria. Transient transfection of neuronal GT1-7 cells with an NR1-NR2a NMDA receptor subunit cassette specifically targeting mitochondria resulted in a significant increase in mitochondrial calcium and neuroprotection against glutamate-induced cell death. Mitochondria prepared from GT1-7 cells in which the NR1 subunit of NMDA receptors was silenced demonstrated a decrease in calcium uptake. Our findings are the first to demonstrate that mitochondria express a calcium transport protein that shares characteristics with the NMDA receptor and may play a neuroprotective role.     

Subunit assembly of N-methyl-d-aspartate receptors analyzed by fluorescence resonance energy transfer

N-methyl-d-aspartate (NMDA) receptors play major roles in synaptic transmission and plasticity, as well as excitotoxicity. NMDA receptors are thought to be tetrameric complexes mainly composed of NMDA receptor (NR)1 and NR2 subunits. The NR1 subunits are required for the formation of functional NMDA receptor channels, whereas the NR2 subunits modify channel properties. Biochemical and functional studies indicate that subunits making up NMDA receptors are organized into a dimer of dimers, and the N termini of the subunits are major determinants for receptor assembling. Here we used a biophysical approach, fluorescence resonance energy transfer, to analyze the assembly of intact, functional NMDA receptors in living cells. The results showed that NR1, NR2A, and NR2B subunits could form homodimers when they were expressed alone in HEK293 cells. Subunit homodimers were also found existing in heteromeric NMDA receptors formed between NR1 and NR2 subunits. These findings are consistent with functional NMDA receptors being arranged as a dimer of dimers. In addition, our data indicated that the conformation of NR1 subunit homodimers was affected by the partner NR2 subunits during the formation of heteromeric receptor complexes, which might underlie the mechanism by which NR2 subunits modify NMDA receptor function.     

Effects of streptozotocin-diabetes on the hippocampal NMDA receptor complex in rats

In animal models of diabetes mellitus, such as the streptozotocin-diabetic rat (STZ-rat), spatial learning impairments develop in parallel with a reduced expression of long-term potentiation (LTP) and enhanced expression of long-term depression (LTD) in the hippocampus. This study examined the time course of the effects of STZ-diabetes and insulin treatment on the hippocampal post-synaptic glutamate N-methyl-D-aspartate (NMDA) receptor complex and other key proteins regulating hippocampal synaptic transmission in the post-synaptic density (PSD) fraction. In addition, the functional properties of the NMDA-receptor complex were examined. One month of STZ-diabetes did not affect the NMDA receptor complex. In contrast, 4 months after induction of diabetes NR2B subunit immunoreactivity, CaMKII and Tyr-dependent phosphorylation of the NR2A/B subunits of the NMDA receptor were reduced and alphaCaMKII autophosphorylation and its association to the NMDA receptor complex were impaired in STZ-rats compared with age-matched controls. Likewise, NMDA currents in hippocampal pyramidal neurones measured by intracellular recording were reduced in STZ-rats. Insulin treatment prevented the reduction in kinase activities, NR2B expression levels, CaMKII-NMDA receptor association and NMDA currents. These findings strengthen the hypothesis that altered post-synaptic glutamatergic transmission is related to deficits in learning and plasticity in this animal model.     

Neural cell adhesion molecule-associated polysialic acid inhibits NR2B-containing N-methyl-D-aspartate receptors and prevents glutamate-induced cell death

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.     

Estradiol induces physical association of neuronal nitric oxide synthase with NMDA receptor and promotes nitric oxide formation via estrogen receptor activation in primary neuronal cultures

Estrogens and nitric oxide (NO) exert wide-ranging effects on brain function. Recent evidence suggested that one important mechanism for the regulation of NO production may reside in the differential coupling of the calcium-activated neuronal NO synthase (nNOS) to glutamate NMDA receptor channels harboring NR2B subunits by the scaffolding protein post-synaptic density-95 (PSD-95), and that estrogens promote the formation of this ternary complex. Here, we demonstrate that 30-min estradiol-treatment triggers the production of NO by physically and functionally coupling NMDA receptors to nNOS in primary neurons of the rat preoptic region in vitro . The ability of estradiol to activate neuronal NO signaling in preoptic neurons and to promote changes in protein-protein interactions is blocked by ICI 182,780, an estrogen receptor antagonist. In addition, blockade of NMDA receptor NR2B subunit activity with ifenprodil or disruption of PSD-95 synthesis in preoptic neurons by treatment with an anti-sense oligodeoxynucleotide inhibited the estradiol-promoted stimulation of NO release in cultured preoptic neurons. Thus, estrogen receptor-mediated stimulation of the nNOS/PSD-95/NMDA receptor complex assembly is likely to be a critical component of the signaling process by which estradiol facilitates coupling of glutamatergic fluxes for NO production in neurons.     

NMDA receptor function: subunit composition versus spatial distribution

NMDA receptors (NMDARs) play a pivotal role in the regulation of neuronal communication and synaptic function in the central nervous system. The subunit composition and compartmental localization of NMDARs in neurons affect channel activity and downstream signaling. This review discusses the distinct NMDAR subtypes and their function at synaptic, perisynaptic, and extrasynaptic sites of excitatory and inhibitory neurons. Many neurons express more than one of the modulatory NR2 subunits that participate in the formation of di- and/or triheteromeric channel assemblies (e.g., NR1/NR2A, NR1/NR2B, and/or NR1/NR2A/NR2B). Depending on the subunit composition and presence or absence of intracellular binding partners along the postsynaptic membrane, these NMDAR subtypes are allocated to distinct synaptic inputs converging onto a neuron or are distributed differentially among synaptic or extrasynaptic sites. These sites can carry NR2A and NR2B subunits, supporting the hypothesis that the spatial distribution of scaffolding and signaling complexes critically determines the full spectrum of NMDAR signaling.The author thanks the Deutsche Forschungsgemeinschaft for financial support (Ko 1064/5).     

Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor

N-methyl-D-aspartate (NMDA) receptors play crucial roles in excitatory synaptic transmission as well as in excitotoxicity. A growing body of evidence suggests that the regulation of both subunit composition and the number of NMDA receptors reaching the surface membrane are tightly regulated. Recently, we have shown that the third membrane domains (M3) of both NR1 and NR2B subunits contain endoplasmic reticulum (ER) retention signals that prevent the unassembled subunits from leaving the ER. Furthermore, these membrane domains together with NR1 M4 are necessary for negating the ER retention signals found in M3 of NR1 and NR2B. In this addendum, we present new electrophysiological data showing that mutation of the HLFY motif, located immediately after M4 of the NR2B subunit, abolishes the surface trafficking of full-length NR1/NR2B complexes (supporting previous immunofluorescent experiments from our lab); however, the deletion of the NR2B C-terminus including the HLFY motif did not affect the formation of functional receptors when two pieces of the NR2B subunit, NR2B truncated before M4 and NR2B M4, were co-expressed together with the NR1 subunit. These observations will help to uncover the processes involved in the assembly of NR1 and NR2 subunits into functional NMDA receptors.     

Molecular Dissection of Native Mammalian Forebrain NMDA Receptors Containing the NR1 C2 Exon: Direct Demonstration of NMDA Receptors Comprising NR1, NR2A, and NR2B Subunits Within the Same Complex

Abstract: The subunit compositions of the NR1 C2 exon-containing N -methyl- d -aspartate (NMDA) receptors of adult mammalian forebrain were determined by using a combination of immunoaffinity chromatography and immunoprecipitation studies with NMDA receptor subunit-specific antibodies. NMDA receptors were solubilised by sodium deoxycholate, pH 9, and purified by anti-NR1 C2 antibody affinity chromatography. The purified receptor subpopulation showed immunoreactivity with anti-NR1 C2, anti-NR1 N1, anti-NR1 C2', anti-NR2A, and anti-NR2B NMDA receptor antibodies. The NR1 C2-receptor subpopulation was subjected to immunoprecipitation using anti-NR2B antibodies and the resultant immune pellets analysed by immunoblotting where anti-NR1 C2, anti-NR1 C2', anti-NR2A, and anti-NR2B immunoreactivities were all found. Quantification of the immunoblots showed that 46% of the NR1 C2 immunoreactivity was associated with the NR2B subunit. Of this, 87% (i.e., 40% of total) were NR1 C2/NR2B receptors and 13% (6% of total) were NR1 C2/NR2A/NR2B, thus identifying the triple combination as a minor receptor subset. These results demonstrate directly, for the first time, the coexistence of the NR2A and NR2B subunits in native NMDA receptors. They show the coexistence of two splice forms of the NR1 subunit, i.e., NR1 C2 and NR1 C2', in native receptors and, in addition, they imply an NMDA receptor subpopulation containing four types of NMDA receptor subunit, NR1 C2, NR1 C2', NR2A, and NR2B, which, in accord with molecular size determinations, predicts that the NMDA receptor is at least tetrameric. These results are the first quantitative study of NMDA receptor subtypes and demonstrate molecular heterogeneity for both the NR1 and the NR2 subunits in native forebrain NMDA receptors.     

Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor

N-methyl-D-aspartate (NMDA) receptors play crucial roles in excitatory synaptic transmission as well as in excitotoxicity. A growing body of evidence suggests that the regulation of both subunit composition and the number of NMDA receptors reaching the surface membrane are tightly regulated. Recently, we have shown that the third membrane domains (M3) of both NR1 and NR2B subunits contain endoplasmic reticulum (ER) retention signals that prevent the unassembled subunits from leaving the ER. Furthermore, these membrane domains together with NR1 M4 are necessary for negating the ER retention signals found in M3 of NR1 and NR2B. In this addendum, we present new electrophysiological data showing that mutation of the HLFY motif, located immediately after M4 of the NR2B subunit, abolishes the surface trafficking of full-length NR1/NR2B complexes (supporting previous immunofluorescent experiments from our lab); however, the deletion of the NR2B C-terminus including the HLFY motif did not affect the formation of functional receptors when two pieces of the NR2B subunit, NR2B truncated before M4 and NR2B M4, were co-expressed together with the NR1 subunit. These observations will help to uncover the processes involved in the assembly of NR1 and NR2 subunits into functional NMDA receptors.     

Chronic ethanol exposure delays the 'developmental switch' of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the 'developmental switch' of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol-treated cells than in control cells. Cross-linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell-surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol-treated cells, as determined by NMDA/glycine-induced increases in intracellular Ca(2+). The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.     

Fyn-mediated phosphorylation of NR2B Tyr-1336 controls calpain-mediated NR2B cleavage in neurons and heterologous systems

Cleavage of the intracellular carboxyl terminus of the N-methyl-d-aspartate (NMDA) receptor 2 subunit (NR2) by calpain regulates NMDA receptor function and localization. Here, we show that Fyn-mediated phosphorylation of NR2B controls calpain-mediated NR2B cleavage. In cultured neurons, calpain-mediated NR2B cleavage is significantly attenuated by blocking NR2B phosphorylation of Tyr-1336, but not Tyr-1472, via inhibition of Src family kinase activity or decreasing Fyn levels by small interfering RNA. In HEK cells, mutation of Tyr-1336 eliminates the potentiating effect of Fyn on calpain-mediated NR2B cleavage. The potentiation of NR2B cleavage by Fyn is limited to cell surface receptors and is associated with calpain translocation to plasma membranes during NMDA receptor activation. Finally, reducing full-length NR2B by calpain does not decrease extrasynaptic NMDA receptor function, and truncated NR1/2B receptors similar to those generated by calpain have electrophysiological properties matching those of wild-type receptors. Thus, the Fyn-controlled regulation of NMDA receptor cleavage by calpain may play critical roles in controlling NMDA receptor properties during synaptic plasticity and excitotoxicity.     

Amyloid precursor protein 695 associates with assembled NR2A‐ and NR2B‐containing NMDA receptors to result in the enhancement of their cell surface delivery

This is a study of the interaction between the two NMDA neurotransmitter receptor subtypes, NR1/NR2A and NR1/NR2B, and amyloid precursor protein (APP) 695, the major APP variant expressed in neurones. APP695 co‐immunoprecipitated with assembled NR1‐1a/NR2A and NR1‐1a/NR2B NMDA receptors following expression in mammalian cells. Single NR1‐1a, NR1‐2a, NR1‐4b^c‐Myc, or NR2 subunit transfections revealed that co‐association of APP695 with assembled NMDA receptors was mediated via the NR1 subunit; it was independent of the NR1 C1, C2, and C2′ cassettes and, the use of an NR1‐2a^c‐Myc‐trafficking mutant suggested that interaction between the two proteins occurs in the endoplasmic reticulum. The use of antibodies directed against extracellular and intracellular NR2 subunit epitopes for immunoprecipitations suggested that APP/NMDA receptor association was mediated via N‐terminal domains. Anti‐APP antibodies immunoprecipitated NR1, NR2A, and NR2B immunoreactive bands from detergent extracts of mammalian brain; reciprocally, anti‐NR1 or anti‐NR2A antibodies co‐immunoprecipitated APP immunoreactivity. Immune pellets from brain were sensitive to endoglycosidase H suggesting that, as for heterologous expression, APP and NMDA receptor association occurs in the endoplasmic reticulum. Co‐expression of APP695 in mammalian cells resulted in enhanced cell surface expression of both NR1‐1a/NR2A and NR1‐1a/NR2B NMDA receptors with no increase in total subunit expression. These findings are further evidence for a role of APP in intracellular trafficking mechanisms. Further, they provide a link between two major brain proteins that have both been implicated in Alzheimer’s disease.