The cloning and characterization of the gene for the fourth subunit of a glutamate-binding protein complex in rat brain synaptic membranes are described. The cloned rat brain cDNA contained two open reading frames (ORFs) encoding 8.9- (PRO1) and 9.5-kDa (PRO2) proteins. The cDNA sequence matched contiguous genomic DNA sequences in rat chromosome 17. Both ORFs were expressed within the structure of a single brain mRNA and antibodies against unique sequences in PRO1- and PRO2-labeled brain neurons in situ, indicative of bicistronic gene expression. Dicistronic vectors in which ORF1 and ORF2 were substituted by either two different fluorescent proteins or two luciferases indicated concurrent, yet independent translation of the two ORFs. Transfection with noncapped mRNA led to cap-independent translation of only ORF2 through an internal ribosome entry sequence preceding ORF2. In vitro or cell expression of the cloned cDNA led to the formation of multimeric protein complexes containing both PRO1 and PRO2. These complexes had low affinity (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)-sensitive phencyclidine-binding sites. Overexpression of PRO1 and PRO2 in CHO cells, but not neuroblastoma cells, caused cell death within 24-48 h. The cytotoxicity was blocked by concurrent treatment with MK-801 or by two tetrahydroisoquinolines that bind to phencyclidine sites in neuronal membranes. Co-expression of two of the other subunits of the protein complex together with PRO1/PRO2 abrogated the cytotoxic effect without altering PRO1/PRO2 protein levels. Thus, this rare mammalian bicistronic gene coded for two tightly interacting brain proteins forming a low affinity phencyclidine-binding entity in a synaptic membrane complex.

Neurons in the hippocampal CA1 region are particularly sensitive to oxidative stress (OS), whereas those in CA3 are resistant. To uncover mechanisms for selective CA1 vulnerability to OS, we treated organotypic hippocampal slices with duroquinone and compared transcriptional profiles of CA1 vs CA3 cells at various intervals. Gene Ontology and Biological Pathway analyses of differentially expressed genes showed that at all time points, CA1 had higher transcriptional activity for stress/inflammatory response, transition metal transport, ferroxidase, and presynaptic signaling activity, while CA3 had higher GABA-signaling, postsynaptic, and calcium and potassium channel activity. Real-time PCR and immunoblots confirmed the transcriptome data and the induction of OS by duroquinone in both hippocampal regions. Our functional genomics approach has identified in CA1 cells molecular pathways as well as unique genes, such as guanosine deaminase, lipocalin 2, synaptotagmin 4, and latrophilin 2, whose time-dependent induction following the initiation of OS may represent attempts at neurite outgrowth, synaptic recovery, and resistance against OS.

Oxidative stress (OS) causes extensive cell death in the CA1 but not the CA3 region of the hippocampus. We found that the CA1 region of hippocampus explants, cultured under normal conditions, had significantly higher superoxide levels and expressed both anti-oxidant genes and genes related to the generation of reactive oxygen species at significantly higher levels than the CA3. These observations were indicative of high intrinsic OS in CA1.

The anti-apoptotic effect of Bcl-2 is well established, but the detailed mechanisms are unknown. In the present study, we show in vitro a direct interaction of Bcl-2 with the rat skeletal muscle SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), leading to destabilization and inactivation of the protein. Recombinant human Bcl-2D21, a truncated form of Bcl-2 with a deletion of 21 residues at the C-terminal membrane-anchoring region, was expressed and affinity-purified as a glutathione S-transferase fusion protein. Bcl-2D21 co-immunoprecipitated and specifically interacted with SERCA in an in vitro-binding assay. The original level of Bcl-2 in sarcoplasmic reticulum vesicles was very low, i.e. hardly detectable by immunoblotting with specific antibodies. The addition of Bcl-2D21 to the sarcoplasmic reticulum resulted in the inhibition of the Ca2+-ATPase activity dependent on the Bcl-2D21/SERCA molar ratio and incubation time. A complete inactivation of SERCA was observed after 2.5 h of incubation at approx. 2:1 molar ratio of Bcl-2D21 to SERCA. In contrast, Bcl-2D21 did not significantly change the activity of the plasma-membrane Ca2+-ATPase. The redox state of the single Cys158 residue in Bcl-2D21 and the presence of GSH did not affect SERCA inhibition. The interaction of Bcl-2D21 with SERCA resulted in a conformational transition of SERCA, assessed through a Bcl-2-dependent increase in SERCA thiols available for the labelling with a fluorescent reagent. This partial unfolding of SERCA did not lead to a higher sensitivity of SERCA towards oxidative inactivation. Our results suggest that the direct interaction of Bcl-2 with SERCA may be involved in the regulation of apoptotic processes in vivo through modulation of cytoplasmic and/or endoplasmic reticulum calcium levels required for the execution of apoptosis.

Both protein and mRNA for the NR1 subunit of N-methyl-d-aspartate receptors are present in neuronal dendrites and undergo changes in distribution following synaptic excitation. However, the expression of all exonic splice variants of NR1 in dendrites has not been determined. In the present study, antibodies against the exon 5 (ex5) peptide sequence labeled proteins mostly in the soma of hippocampus neurons, whereas antibodies against ex21 or ex22 labeled cell bodies and dendrites. Antisense cRNAs for ex5 hybridized with mRNAs in cell bodies, whereas cRNAs for ex21 with mRNAs in both cell bodies and dendrites. Antisense DNA to a 24-base sequence identified as being present only in the 5′-UTR of cDNAs lacking ex5 (ex5−), hybridized with mRNAs in soma and dendrites and this labeling was coincident, mostly, with RNA granules. Insertion of the 24-base DNA ahead of that for enhanced green fluorescent protein (EGFP) increased the transport of EGFP mRNA and the expression of EGFP in neurites of neurons in culture. Fluorescent sense mRNA that contained the 24-base sequence bound to proteins in dendrites and to two proteins, 60 and 70 kDa, in brain microsomes. Proteins of similar size were also labeled by [32P]CTP-mRNA for NR1-1a, which contains the 24-base 5′-UTR sequence, but not for NR1-2b, which does not. Biotinylated 24-base sense mRNA was used to purify from brain microsomes two RNA-binding proteins (60 and 70 kDa). We concluded that the 24-base sequence in 5′-UTR of ex5− mRNA functioned as a cis-acting, dendrite-targeting element recognized selectively by two microsome proteins.

Excessive superoxide (O(-)(2)) formation is toxic to cells and organisms. O(-)(2) reacts with either iron-sulfur centers or cysteines (Cys) of cytoplasmic proteins. Reactions with membrane proteins, however, have not been fully characterized. In the present studies, the reaction of O(-)(2) with a protein complex that has glutamate/N-methyl-D-aspartate (NMDA) receptor characteristics and with one of the subunits of this complex was examined. Exposure of the complex purified from neuronal membranes and the recombinant glutamate-binding protein (GBP) subunit of this complex to the O(-)(2)-generating system of xanthine (X) plus xanthine oxidase (XO) caused strong inhibition of L-[3H]glutamate binding. Inhibition of glutamate binding to the complex and GBP by O(-)(2) was greater than that produced by H(2)O(2), another product of the X plus XO reaction. Mutation of two cysteine (Cys) residues in recombinant GBP (Cys(190,191)) eliminated the effect of O(-)(2) on L-[3H]glutamate binding. Both S-thiolation reaction of GBP in synaptic membranes with [35S]cystine and reaction of Cys residues in GBP with [3H]NEM were significantly decreased after exposure of membranes to O(-)(2). Inhibition of cysteylation of membrane GBP by O(-)(2) was still observed after iron chelation by desferrioxamine, albeit diminished, and was not altered by the presence of catalase. Overall, the results indicated that GBP exposure to O(-)(2) modified Cys residues in this protein. The modification was not characterized but it was probably that of disulfide formation.

A group of four proteins with recognition sites for L-glutamate, N-methyl-D-aspartate, glycine, and competitive and non-competitive inhibitors of N-methyl-D-aspartate receptors was previously purified from rat brain synaptic membranes. The biochemical and immunochemical characteristics of this complex, as well as the sequences of the complementary DNAs of three subunits, are distinct from those of other glutamate receptors, transporters, or enzymes. The function of this complex has not yet been defined, but it appears to be involved in glutamate-induced neuronal excitation and toxicity. It is not known whether all protein components of the complex are expressed in the same populations of brain cells. In the present study, immunohistochemical and in situ hybridization were used to map the distribution of the glutamate-binding, glycine/thienylcyclohexylpiperidine-binding, and carboxypiperazinyl-propylphosphonate-binding protein subunits of the complex. These proteins were abundantly expressed in pyramidal neurons of the hippocampus and cerebral cortex, and in granule cells of the dentate gyrus, cerebellum, and olfactory tubercle. Based on these results, it was concluded that the three subunits of the complex have similar patterns of expression in rat brain. The distribution of one subunit of the complex, glutamate-binding protein, was traced throughout the rat brain, thus providing a potential map of the expression of the complex in rodent brain. In addition, probes were developed in the present study that should be useful in future explorations of the role of these proteins in brain function and of the possible co-localization of the protein subunits in single cells or cell processes.

A complex of four proteins isolated from neuronal membranes has ligand binding sites forN-methyl-d-aspartate (NMDA) receptor agonists and antagonists and forms NMDA-activated ion channels upon reconstitution into lipid membranes. In this study, the cDNA of a subunit of this complex containing binding sites for the competitive antagonists of NMDA receptors was cloned. The cDNA clone coded for a protein of 719 amino acids (78.9 kDa). The expressed protein had binding activity for the agonistsl-[3H]glutamate and [3H]glycine, the antagonist (±)-[3H]-(E)-2-amino-4-propyl-5-phosphonopentanoic acid ([3H]CGP 39653), but not the ion channel inhibitors. The cloned cDNA had no homology to other cloned cDNAs. Northern blot analyses indicated high expression of an 3.8 kb poly(A+) RNA in brain, but not in other tissues. These findings indicate that proteins that have recognition sites for NMDA receptor activators and inhibitors and that differ from the well-characterized NMDA receptor proteins NR1-3 are expressed in mammalian brain.

The functional reconstitution of glutamate receptor proteins purified from mammalian brain has been difficult to accomplish. However, channels activated by L-glutamate (L-Glu) and N-methyl-D-aspartate (NMDA) were detected in planar lipid bilayer membranes (PLMs) following the reconstitution of a complex of proteins with binding sites for NMDA receptor (NMDAR) ligands. The presence of glycine was necessary for optimal activation. A linear current-voltage relationship was observed with the reversal potential being zero. Channels activated by L-Glu had conductances of 23, 47 and 65 pS, and were suppressed partially by competitive and fully by noncompetitive inhibitors of NMDARs. Magnesium had little effect on the reconstituted channels.

Antibodies raised against a 43 kDa component of a complex of synaptic membrane proteins with ligand binding sites characteristic of glutamate/N-methyl-d-aspartate (NMDA) receptors, were used previously to clone a cDNA for a glycine-, glutamate-, and thienylcyclohexylpirperidine (TCP)-binding protein, pGlyBP (Kumar et al., Biochem. Biophys. Res. Commun.216, 390–398, 1995). In the present studies, the antibodies were shown to label a 60 kDa protein, in synaptic membranes, that was relatively hydrophilic as demonstrated by its predominant separation in the detergent-depleted phase of proteins solubilized with Triton X-114. A 55–60 kDa protein was purified from rat brain synaptic membranes by chromatographic separation through matrices derivatized with 5,7-di-chlorokynurenic acid (5,7-DCK) followed by chromatography on a matrix derivatized with 8-hydroxyquinoline (8-OHQ). The isolated fractions were highly enriched in strychnine-insensitive [3H]glycine, NMDA- and glutamate-sensitive l-[3H]glutamate, and MK-801-sensitive [3H]TCP binding sites. The purified protein bound [3H]glycine with a stoichiometry of 1.1-1.2 mol glycine per mol protein and exhibited both high (KD = 280 nM) and low affinity (KD = 30 μM) glycine binding sites. Glycine binding was inhibited by d-serine and R-(+)-3-amino-1-hydroxypyrrolidin-2-one (R-(+)-HA-966). The KD values for high and low affinity sites of glycine binding as well as those for the inhibition by R-(+)-HA-966 were very similar to the KDs for glycine binding to the expressed pGlyBP. Both l-glutamate and glycine activated [3H]TCP binding to the isolated proteins, but with relatively low affinity. The anti-43 kDa antibodies reacted strongly with the 55–60 kDa protein. Based on these results, it appears that the 60 kDa glycoprotein in brain synaptic membranes described in the present study is the same protein as the cloned pGlyBP.

Abstract: N‐Methyl‐d‐asparate receptors (NMDARs) are a major target of ethanol effects in the nervous system. Haloperidol‐insensitive, but dizocilpine (MK‐801)‐sensitive, binding of N‐[1‐(2‐[3H]thienyl)cyclohexyl]piperidine ([3H]TCP) to synaptic membranes has the characteristics of ligand interaction with the ion channel of NMDARs. In the present studies, ethanol produced a concentration‐dependent decrease in the maximal activation of [3H]TCP binding to synaptic membranes by NMDA and Gly, but a moderate change in the activation by l‐Glu when l‐Glu was present at concentrations < 100 µM. However, ethanol (100 mM) inhibited completely the activation of [3H]TCP binding produced by high concentrations of l‐Glu (200–400 µM). It also inhibited strongly the activation of [3H]TCP binding by spermidine or spermidine plus Gly. In a purified complex of proteins that has l‐Glu‐, Gly‐, and [3H]TCP‐binding sites, ethanol (100 mM) decreased significantly the maximal activation of [3H]TCP binding produced by either l‐Glu or Gly. Activation constants (Kact) for l‐Glu and Gly acting on the purified complex were 12 and 28 µM, respectively. Ethanol had no significant effect on the Kact of l‐Glu but caused an increase in the Kact of Gly. These studies have identified at least one protein complex in neuronal membranes whose response to both l‐Glu and Gly is inhibited by ethanol. These findings may explain some of the effects of acute and chronic ethanol treatment on the function and expression of the subunits of this complex in brain neurons.

Besides the N-methyl-D-aspartate (NMDA) receptor proteins NR1 and NR2, another complex of proteins which has been shown to contain ligand-binding sites characteristic of NMDA receptors is expressed in cerebellar granule cells. One of the proteins in the latter complex is the 71 kDa glutamate-binding protein (GBP). To determine the role of the GBP in the response to NMDA, primary cultures of cerebellar granule cells were treated with an antisense oligonucleotide complementary to mRNA for this protein. This treatment substantially reduced both mRNA and protein levels of the GBP, as well as the response of the cells to NMDA, measured as an increase in intracellular Ca2+ with fura-2 fluorescence. The antisense oligonucleotide treatment did not alter the Ca2+ responses to KC1 or kainate. Chronic ethanol exposure has previously been shown to increase NMDA receptor function and the density of binding sites for the NMDA receptor channel blocker, dizocilpine, in cerebellar granule cells. Chronic exposure of the cells to 100mM ethanol is now shown to result in significant increases in mRNA and protein levels for the GBP (45% and 100%, respectively). Ethanol treatment did not affect mRNA levels for NR1 or NR2A, caused only a small increase (20%) in protein levels for NR1, and resulted in a decrease (30%) in NR2A protein. Although a role of the NMDA receptor NR1/NR2 subunits cannot be ruled out, these results are compatible with the hypothesis of involvement of the GBP in the chronic ethanol-induced increase in NMDA receptor function in cerebellar granule cells.

In response to biochemical factors like catecholamines, bradykinins, histamine and physical factors like shear stress, endothelial cells release a non prostanoid factor, called endothelium derived relaxing factor (EDRF), which relaxes vascular smooth muscle. Since this discovery in 1980, many additional agents have been shown to stimulate release of EDRF from endothelium. Biological and chemical evidence has supported the proposal that EDRF is actually nitric oxide (NO). Research on the synthesis, inhibition and physiological roles of nitric oxide (NO) has led to studies of its involvement in blood pressure homeostasis and immune functions.

Polyclonal antibodies (Ab′s) were raised against a 43-kDa component of a protein complex that has ligand recognition sites similar to those of brain N-methyl-D-aspartate (NMDA) receptors. The Ab′s were used to immunopurify from brain synaptic membranes a 60-kDa glycine (Gly), glutamate (Glu) and thienylcyclohexylpiperidine (TCP)-binding protein and to screen a rat hippocampal cDNA expression library. A 1.85-kb clone, pGlyBP, coding for a protein of 470 amino acids (52.7 kDa) was identified. Northern blot analyses performed on poly(A+) RNA from brain revealed hybridization of the labeled cDNA probes to transcripts of 1.9 kb. E. coli transformed with the pGyBP expressed a protein that was recognized by the anti-43 kDa Ab′s and had recognition sites for Gly, Glu and TCP. The cloned protein has 2 glycosylation sites, 3 hydrophobic domains, 4 cysteine-rich motifs (C-X2-C-X16-20C-X5-11), and 2 regions homologous to the NR1 receptor protein.

Kumar, K N; Babcock, K K; Johnson, P S; Chen, X; Riegel, A; Michaelis, E K Author Information

A method was developed for the solubilization of approximately 50% of proteins in synaptic membranes that have ligand-binding characteristics of N-methyl-D-aspartate (NMDA) receptors. Affinity chromatographic separation of the solubilized proteins through L-glutamate-derivatized matrices and subsequent elution by NMDA-containing buffers led to the purification of four predominant proteins with estimated sizes of 67-70, 53-62, 41-43, and 28-36 kDa. The co-purification of NMDA-sensitive L-glutamate binding, dizocilpine-sensitive thienylcyclohexyl piperidine (TCP)-binding, and strychnine-insensitive glycine-binding proteins was achieved by this affinity chromatographic procedure. Glutamate, glycine, and the polyamine spermidine increased both the on rate and the equilibrium level of [3H]TCP binding to the isolated proteins. The group of proteins eluted by NMDA from the glutamate-derivatized matrices could be further purified through size exclusion chromatography of the NMDA-sensitive glutamate-binding from the dizocilpine-sensitive TCP-binding proteins. Polyclonal and monoclonal antibodies to the cloned NMDA receptor protein NMDAR1 did not react with any proteins in the solubilized membrane proteins or the purified fractions. However, immunoreaction of antibodies raised against a glutamate-binding protein and a phosphonoaminocarboxylic acid-binding protein indicated that these are two of the major proteins in the purified fractions. These studies indicate that these two proteins might be components of a complex that has some of the characteristics of NMDA receptors and that neuronal membranes may contain varieties of NMDA-like receptors composed of protein subunits that differ from the NMDAR1 and NMDAR2 receptor proteins.