Cloning and structure of the gene encoding the human N-methyl-d-aspartate receptor (NMDAR1) (original) (raw)
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Cloning and expression of the human N-methyl-d-aspartate receptor subunit NR3A
Neuroscience Letters, 2001
Native N-methyl-d-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding to a 93% homology at the amino acid level. Based on available data from human genome databases, we localized the gene to chromosome 9. The transcript could be detected by in situ hybridization in human fetal spinal cord and forebrain. Two splice variants of NR3A have been reported in rat brain, the longer of the two containing a 60 bp insert in the intracellular domain. We were unable to detect this 60 bp insert in fetal or adult human brain, suggesting that only the short variant is expressed in humans. q
Gene, 2002
The 5 0 flanking region and the 5 0 untranslated region (5 0 UTR) of the rat N-methyl-d-aspartate receptor subunit 2A were cloned and sequenced using polymerase chain reaction-mediated chromosome walking. The complementary DNA (cDNA) was obtained by rapid amplification of 5 0 cDNA ends (5 0 RACE). The comparison of the cDNA and the genomic sequences showed that the 5 0 UTR contained two introns and three exons, the third exon overlapping the beginning of the coding region. Transcriptional initiation sites were identified by 5 0 RACE and RNA-protection assays, using total rat brain RNA. The main start sites were found at 2591, 2577, 2560 and 2541 nucleotides 5 0 of the AUG. The promoter region lacked TATA and CAAT positioning elements. A CpG island of about 700 bp overlapped the 5 0 flanking sequences and the 5 0 UTR. The CpG island was inside a wider GC-rich region (66% GC) spanning the entire 5 0 UTR. Comparison of the rat sequences with the human sequences from the Human Genome Data Bank revealed that the 5 0 UTR exon 2 was extremely conserved with 95.8% sequence identity, as were the initial 640 bp of 5 0 flanking sequences, with 78% sequence identity. Beyond this point, sequence identity dropped abruptly to 44%. Putative recognition sequences for the transcription factors S8, Sp1, GATA, AML1 and NF-kB were identified in both the rat and human promoter sequences. q Abbreviations: 5 0 UTR, 5 0 untranslated region; bp, base pair(s); CNS, central nervous system; MCS, multiple cloning sites; NMDA, N-methyl-daspartate; NR, N-methyl-d-aspartate receptor subunit; 5 0 RACE, rapid amplification of 5 0 cDNA ends
Neuroscience, 1996
The expression of the messenger RNAs encoding N-methyl-D-aspartate receptor subunits in neurologically normal post-mortem human brain was studied by in situ hybridization. In the caudate, putamen and nucleus accumbens strong hybridization signals were observed for N-methyl-D-aspartate RI-1 messenger RNA but much weaker signals for N-methyl-o-aspartate R1-3 and N-methyl-D-aspartate R1-4. N-Methyl-D-aspartate RI-2 was not detectable. N-methyl-D-aspartate R2B was the only N-methyl-D-aspartate R2 subunit detected in these nuclei. In the hippocampus the messenger RNAs for both N-methyl-D-aspartate R1 1 and N-methyl-D-aspartate R1 4 were strongly expressed in the dentate gyrus, CA343A 1 pyramidal cells, subiculum, entorhinal cortex and perirhinal cortex. Much lower expression was seen for N-methyl-n-aspartate RI~ and N-methyl-D-aspartate R1-3. The messenger RNAs for both N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2B, but not N-methyl-D-aspartate R2C, subunits were expressed in the hippocampus. In the temporal cortex all N-methyl-D-aspartate R1 isoforms were expressed (N-methyl-D-aspartate RI-1 and N-methyl-D-aspartate R1-4 being the most abundant) and N-methyl-D-aspartate R2A and N-methyl-o-aspartate R2B but not N-methyl-D-aspartate R2C were also moderately expressed. In the brain stem N-methyl-D-aspartate R1 4 was strongly expressed in various nuclei including the locus coeruleus, nucleus centralis superior and deep pontine nuclei. Only weak expression was seen for N-methyl-D-aspartate RI-1 and N-methyl-D-aspartate R1-3 but not N-methyl-Daspartate RI-2; of the N-methyl-D-aspartate R2 subunits only N-methyl-D-aspartate R2C was found to be expressed in these nuclei. In the cerebellum all the N-methyl-D-aspartate 1 isoforms were expressed (mostly N-methyl-D-aspartate RI-4) in the Purkinje layer which also expressed N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2C. In the molecular layer cells were found expressing N-methyl-D-aspartate RI-4 and N-methyl-D-aspartate R2B and cells in the granule layer were found to express N-methyl-D-aspartate RI-1, N-methyl-D-aspartate R1 3 and N-methyl-D-aspartate R1-4 and N-methyl-D-aspartate R2C only. Preliminary studies indicated that the messenger RNA for the N-methyl-D-aspartate R2D subunit was not expressed in the above areas of brain.
Journal of Neurochemistry, 2002
cDNAs encoding four isoforms of the human NMDA receptor (NMDAR) NMDAR2C (hNR2C-1, -2, -3, and -4) have been isolated and characterized. The overall identity of the deduced amino acid sequences of human and rat NR2C-1 is 89.0%. The sequences of the rat and human carboxyl termini (G1y 925-Va11236) are encoded by different exons and are only 71.5% homologous. In situ hybridization in human brain revealed the expression of the NR2C mRNA in the pontine reticular formation and lack of expression in substantia nigra pars compacta in contrast to the distribution pattern observed previously in rodent brain. The pharmacological properties of hNR1A/2C were determined by measuring agonist-induced inward currents in Xenopus oocytes and compared with those of other human NMDAR subtypes. Glycine, glutamate, and NMDA each discriminated between hNR1A/2C-1 and at least one of hNR1A/2A, hNR1A/2B, or hNR1A/2D subtypes. Among the antagonists tested, CGS 19755 did not significantly discriminate between any of the four subtypes, whereas 5,7-dichlorokynurenic acid distinguished between hNR1A/2C and hNR1A/2D. Immunoblot analysis of membranes isolated from HEK293 cells transiently transfected with cDNAs encoding hNR1A and each of the four NR2C isoforms indicated the formation of heteromeric complexes between hNR1A and all four hNR2C isoforms. HEK293 cells expressing hNR1A/ 2C-3 or hNR1A/2C-4 did not display agonist responses. In contrast, we observed an agonist-induced elevation of intracellular free calcium and whole-cell currents in cells expressing hNR1A/2C-1 or hNR1A/2C-2. There were no detectable differences in the macroscopic biophysical properties of hNR1A/2C-1 or hNR1A/2C-2.
Relationship between N-Methyl-D-aspartate Receptor NR1 Splice Variants and NR2 Subunits
Journal of Biological Chemistry, 1996
The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor is assembled from one NR1 subunit, expressed in eight splice variants, and four NR2 subunits (NR2A-D). The combination of subunits and splice variants determines the pharmacological and physiological properties of the receptor. In the present study we investigated the relationship between NR1 splice variants and NR2 subunits in rat brain using a series of antibodies selective for the four NR1 cassettes, which vary in the NR1 splice variants, and for NR2A and NR2B. Sodium deoxycholate at pH 9.0 solubilized about 35% of the receptor, which was intact based on co-immunoprecipitation of NR1 and NR2 subunits and chemical cross-linking of the solubilized receptor. The crosslinked product contained three high molecular weight components, M r ؍ 603,000, 700,000, and 750,000, which were immunolabeled with antibodies to NR1 and to NR2 subunits. Immunoprecipitation analyses using antibodies selective for NR2A and NR2B showed no preferential assembly between NR2 subunits and NR1 splice variants. There was little co-immunoprecipitation of NR2A and NR2B, suggesting that most NMDA receptor complexes contain only one of these subunits. However, receptor complexes can contain at least two different NR1 splice variants. In developing conditions for the solubilization of intact NMDA receptor complexes, we observed a differential solubilization of NR1 and NR2 subunits. NR2 was nearly insoluble in Triton X-100 in both microsomal and synaptic membrane fractions, while NR1 was readily soluble in the microsomal fraction but insoluble in the synaptic membrane fraction. These results suggest that the NR1 subunit is modified when it is incorporated into the synaptic membrane, possibly by strengthening its interaction with NR2 or another synaptic protein.
Gene, 2002
The 5 0 flanking region and the 5 0 untranslated region (5 0 UTR) of the rat N-methyl-d-aspartate receptor subunit 2A were cloned and sequenced using polymerase chain reaction-mediated chromosome walking. The complementary DNA (cDNA) was obtained by rapid amplification of 5 0 cDNA ends (5 0 RACE). The comparison of the cDNA and the genomic sequences showed that the 5 0 UTR contained two introns and three exons, the third exon overlapping the beginning of the coding region. Transcriptional initiation sites were identified by 5 0 RACE and RNA-protection assays, using total rat brain RNA. The main start sites were found at 2591, 2577, 2560 and 2541 nucleotides 5 0 of the AUG. The promoter region lacked TATA and CAAT positioning elements. A CpG island of about 700 bp overlapped the 5 0 flanking sequences and the 5 0 UTR. The CpG island was inside a wider GC-rich region (66% GC) spanning the entire 5 0 UTR. Comparison of the rat sequences with the human sequences from the Human Genome Data Bank revealed that the 5 0 UTR exon 2 was extremely conserved with 95.8% sequence identity, as were the initial 640 bp of 5 0 flanking sequences, with 78% sequence identity. Beyond this point, sequence identity dropped abruptly to 44%. Putative recognition sequences for the transcription factors S8, Sp1, GATA, AML1 and NF-kB were identified in both the rat and human promoter sequences. q Abbreviations: 5 0 UTR, 5 0 untranslated region; bp, base pair(s); CNS, central nervous system; MCS, multiple cloning sites; NMDA, N-methyl-daspartate; NR, N-methyl-d-aspartate receptor subunit; 5 0 RACE, rapid amplification of 5 0 cDNA ends
mRNA distribution in adult human brain of GRIN2B, a N-methyl-d-aspartate (NMDA) receptor subunit
Neuroscience Letters, 1997
The expression of the N-methyl-D-aspartate (NMDA) receptor subunit NR2B/e2 (GRIN2B) in the human adult brain was assayed by in situ hybridisation, by using a specific cRNA probe. The full length GRIN2B cDNA was cloned and sequenced. It showed a 90% nucleotide conservation when compared to the rodent homologue. GRIN2B gene is expressed at high levels in the fronto-parieto-temporal cortex and hippocampus pyramidal cells and, at a lower extent, in the basal ganglia (amygdala and striatum). The cerebellar granule cells does not show any mRNA expression. The non-ubiquitous anatomical distribution of the GRIN2B mRNA in the central nervous system suggests that the gene could be involved in specific functions pertaining to the expressing cell groups.
The Protein Journal, 2006
The N-methyl-D-aspartate (NMDA) receptor subunit NR1 gene can produce eight isoforms in rat brain. A novel methodology for purifying NMDA receptor NR1 subunit from rat brain is reported here using chicken polyclonal antibodies (IgYs) against synthetic peptides corresponding to N1, C1 and C2¢ cassettes. The isolated protein was recognized by produced IgYs and commercial anti-NR1 IgGs, shown by MALDI-TOF MS a MW=131,192 Da (glycosylated form); the enzymatically deglycosylated protein revealed a MW=102,754 Da. The NMDA receptor NR1 subunit was characterized as being a heavily N-glycosylated protein. The isoelectric point was determined (6.3) as being different from that predicted for any of the isoforms (7.9-9.02). Attempts to separate the isoforms from the purified NR1 were unsuccessful, indicating the presence of just one isoform (NR1 111 ). Immunohistochemistry on hippocampus regions CA1, CA3 and Dentate gyrus with anti-N1, anti-N2 and anti-C2¢ IgYs showed different staining intensity, depending upon the antibody assayed.
Revista Brasileira de Zootecnia, 2014
The objective of the present study was to report the molecular cloning and determination of the sequences N-methyl-D-aspartate subunit-1 receptor (NMDAR-1) in the hypothalamus of sheep. The re-amplified DNA template for NMDA-1 from the hypothalamus of male sheep was cloned using pBluescript-Sk-plasmids (pBSK, 2958 bp). Purified plasmids containing the NMDA receptor c-DNA were sequenced using the dye-terminator chemistry at the University of Nebraska-Lincoln, Nebraska, USA. Results were entered into the National Center for Biotechnology Web site as accession number AY434689. The sequence of Ovis aries (sheep) NMDAR-1 mRNA from hypothalamus of male sheep has 94-97% homology with Homo sapiens, 94-100% homology with Sus scrofa, and 88-90% homology with Rattus norvegicus NMDAR-1 mRNA sequence, and 94-97% homology with Homo sapiens glutamate receptor and transcript variant NR1-2 and NR1-1 mRNA sequence. These results show high evolutionary conservation of NMDA receptor subunit-1 across species.