Cloning and sequence analysis of cDNAs encoding human hippocampus N-methyl-d-aspartate receptor subunits: evidence for alternative RNA splicing

doi:10.1016/0378-1119(93)90309-Q

Gene

Volume 131, Issue 2, 15 September 1993, Pages 293-298

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Abstract

Several cDNA clones encoding human $N- methyl- d -aspartate$ receptor (hNRl) subunit polypeptides were isolated from a human hippocampus library. Degenerate oligodeoxyribonucleotide (oligo) primers based on the published rat NR1 (rNR1) amino acid (aa) sequence [K. Moriyoshi et al. Nature 354 (1991) 31–37] amplified a 0.7-kb fragment from a human hippocampus cDNA library, via the polymerase chain reaction (PCR). This fragment was used as a probe for subsequent hybridization screening. DNA sequence analysis of 28 plaque-purified clones indicated three distinct classes, designated hNR1-1, hNR1-2 and hNR1-3, presumably generated by alternative RNA splicing. One of these clones, hNR1-1(5A), was isolated as a full-length cDNA. The hNR1-2 and hNR1-3 cDNAs represented 66.8 and 98.9%, respectively, of the total aa coding information predicted for the polypeptides. The hNR1 cDNAs demonstrated an 84–90.8% nucleotide (nt) identity with the corresponding rodent cDNAs. The nt sequences of hNR1-1, hNR1-2 and hNR1-3 would encode 885-, 901- and 938-aa proteins, respectively, that have 99.1–99.8% identity with the corresponding rodent NR1 (roNRl) subunits. The changes between the predicted aa sequences of hNRl and the corresponding roNRl subunits are confined to the extracellular N-terminal regions. We have also identified two possible allelic variations of the hNR1-3 cDNA that result in aa substitutions in the extracellular N- and C-terminal regions. One of these naturally occurring aa variations is situated within a potential glutamate-binding site.

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Although NR1 subunits can assemble as homomeric ion channels, the current generated by such receptor homomers is only a small fraction of that generated by heteromeric assemblies of NR1 and at least one of the four NR2 subunits (NR2A–NR2D) or NR3 subunits (NR3A–NR3C) (Stephenson, 2001; Paoletti, 2011). NR1 is the essential subunit for a functional NMDA receptor, ubiquitously expressed in the retina (Foldes et al., 1993). It has been shown that every neuron in the ganglion cell layer expresses the NR1 subunit regardless of developmental age in the rat retina.
N-methyl-d-aspartate receptors (NMDARs) belong to the ionotropic glutamate receptors, which play key roles in neuronal communication in the retina. NMDA receptors are tetrameric protein complexes usually comprising two obligatory NMDA receptor 1 (NR1) subunits and modulatory NMDA receptor 2/3 (NR2/3) subunits. Although the expression patterns of different NMDA receptor subunits have been extensively studied, in this study we focused on NR1 protein expression in the rat retina by immunofluorescence double labeling. We show that NR1 labeling is diffusely distributed in the outer plexiform layer (OPL) and throughout the whole inner plexiform layer (IPL). The NR1-immunoreactivity (IR) was displayed in a variety of cells in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Interestingly, NR1 was expressed in both rod and cone bipolar cells identified by specific bipolar cell markers Chx10, protein kinase C (PKC) and recoverin. All the amacrine cells that we studied, including cholinergic, dopaminergic, GABAergic and glycinergic amacrine cells, were NR1-IR positive. In the ganglion cell layer, NR1-IR was expressed in all cells that were positive for the ganglion cell marker Brn3a. Our study suggests that the NR1 subunit is expressed more widely than was previously appreciated.
Molecular pharmacology of human NMDA receptors
2012, Neurochemistry International
Citation Excerpt :
Only few differences are located in the agonist binding and the transmembrane domains, suggesting conservation of key conformational changes that lead to channel gating in response to agonist binding. More detailed analyses of cDNAs and deduced amino acid sequences for human NMDA receptor subunits are described in the original cloning papers (Collins et al., 1993; Daggett et al., 1998; Foldes et al., 1993, 1994a,b; Hess et al., 1996, 1998; Karp et al., 1993; Le Bourdelles et al., 1994; Lin et al., 1996; Planells-Cases et al., 1993). The intracellular CTD varies greatly among different ionotropic glutamate receptor subunits.
N-methyl-d-aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission. NMDA receptors are also important drug targets that are implicated in a number of pathophysiological conditions. To facilitate the transition from lead compounds in pre-clinical animal models to drug candidates for human use, it is important to establish whether NMDA receptor ligands have similar properties at rodent and human NMDA receptors. Here, we compare amino acid sequences for human and rat NMDA receptor subunits and discuss inter-species variation in the context of our current knowledge of the relationship between NMDA receptor structure and function. We summarize studies on the biophysical properties of human NMDA receptors and compare these properties to those of rat orthologs. Finally, we provide a comprehensive pharmacological characterization that allows side-by-side comparison of agonists, un-competitive antagonists, GluN2B-selective non-competitive antagonists, and GluN2C/D-selective modulators at recombinant human and rat NMDA receptors. The evaluation of biophysical properties and pharmacological probes acting at different sites on the receptor suggest that the binding sites and conformational changes leading to channel gating in response to agonist binding are highly conserved between human and rat NMDA receptors. In summary, the results of this study suggest that no major detectable differences exist in the pharmacological and functional properties of human and rat NMDA receptors.
Cloning and expression of the human NMDA receptor subunit NR3B in the adult human hippocampus
2005, Neuroscience Letters
The mammalian genome encodes seven different NMDA receptor subunits. All of these subunits have been cloned in the human except for NR3B. Here, we have successfully obtained two full-length clones of human NR3B using a PCR-based cloning approach. The open reading frame of the consensus sequence contains 3129 nucleotides translating into 1043 amino acids. The overall polypeptide sequence identity with mouse NR3B is 74.9%, which is lower than for the other six NMDA receptor subunits. In particular, the translated part of exon 9 is only 37.8% identical between human and mouse. The GRIN3B gene, which encodes human NR3B, maps to chromosome 19p13.3, between WDR18 and C19orf6 (membralin). Human NR3B is encoded by nine exons, as in mouse NR3B, and exon–intron boundaries are conserved between the species. However, exon 9 is substantially longer in the human. In situ hybridization data shows that NR3B mRNA is expressed in the human hippocampal formation (CA1–CA4 and dentate gyrus) and adjacent neocortex. The expression of NR3A mRNA was restricted to the dentate gyrus and layers IV and V of the neocortex. Our results may have implications for the understanding of the role of NMDA receptors for physiological and pathological processes in these forebrain regions.
N-methyl-D-aspartate receptor subunit NR2A and NR2B messenger RNA levels are altered in the hippocampus and entorhinal cortex in Alzheimer's disease
2002, Journal of the Neurological Sciences
The N-methyl-d-aspartate (NMDA) receptor is a subtype of ionotropic glutamate receptor that is involved in synaptic mechanisms of learning and memory, and mediates excitotoxic neuronal injury. In this study, we tested the hypothesis that NMDA receptor subunit gene expression is altered in Alzheimer's disease (AD), especially in brain regions known to be important in memory. Quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) was used to determine the messenger RNA (mRNA) levels of the NMDA receptor subunits NR1, NR2A, and NR2B in the hippocampus and entorhinal cortex of postmortem brain samples from nine clinically well-characterized AD patients and nine aged controls. Cerebellum, a site minimally affected by AD, was also chosen for comparison assessment. Results showed decreased levels of the NR2 mRNAs in AD brains compared to controls. Reductions of NR2A (46.2%, p<0.01) and NR2B (43.2%, p<0.0001) mRNA levels were identified in the entorhinal cortex. Reductions of NR2A (41.4%, p<0.05) and NR2B (40.6%, p=0.058) mRNA levels were found in the hippocampus. NR1 mRNA levels were similar in all three brain regions in both AD and controls. No significant changes of subunit NR2A and NR2B mRNA levels were identified in the cerebellum. Postmortem delay (PMD), tissue storage time, brain weight, or age of the subjects did not affect these changes. These data suggest that alterations in NMDA receptor subunits, especially the NR2A and NR2B, may be important in AD, particularly in neuronal populations that underlie impaired learning and memory.
Nucleotide sequence, genomic organization, and chromosomal localization of genes encoding the human NMDA receptor subunits NR3A and NR3B
2001, Genomics
The N-methyl- $D$ -aspartate (NMDA) receptors are glutamate-regulated ion channels that are critically involved in important physiological and pathological functions of the mammalian central nervous system. We have identified and characterized the gene encoding the human NMDA receptor subunit NR3A (GRIN3A), as well as the gene (GRIN3B) encoding an entirely novel subunit that we named NR3B, as it is most closely related to NR3A (57.4% identity). GRIN3A localizes to chromosome 9q34, in the region 13–34, and consists of nine coding exons. The deduced protein contains 1115 amino acids and shows 92.7% identity to rat NR3A. GRIN3B localizes to chromosome 19p13.3 and contains, as does the mouse NR3B gene (Grin3b), eight coding exons. The deduced proteins of human and mouse NR3B contain 901 and 900 amino acid residues, respectively (81.6% identity). In situ hybridization shows a widespread distribution of Grin3b mRNA in the brain of the adult rat.
Effects of post-mortem delay on subunits of ionotropic glutamate receptors in human brain
2000, Molecular Brain Research
The effect of post-mortem delay on the stability of the protein subunits that combine to form NMDA and AMPA type glutamate receptors has been assessed in samples of human brain tissue. While most of the subunits (i.e. GluR1, GluR2/3, GluR4, NR1) appear to be stable for up to 18 h post-mortem, the NR2A and NR2B subunits appear to be proteolyzed rapidly following death. These results are consistent with the concept that the proteolytic products of NR2A and NR2B, although at smaller molecular sizes than the full-length protein, are all identifiable on Western blots. Thus, a method is proposed that allows for the estimation of the levels of these labile proteins even in samples obtained up to 18 h post-mortem. Using this method we have estimated the levels of all AMPA and NMDA receptor subunits in selected (i.e. hippocampus, frontal and entorhinal cortex) brain tissue samples obtained from control patients and patients who have died with Alzheimer's disease. Modest decreases in NMDA receptor subunits NR1, NR2A, and NR2B were found in the hippocampus and in frontal cortex while little or no change in any of these subunits were documented in entorhinal cortex. Subunits for AMPA receptors (GluR1, GluR2/3, and GluR4) appeared to show a generalized decrease in all these tissues. As a surrogate marker for overall decreases due to generalized neuronal cell death, levels of neuron-specific enolase were measured in all tissues and were found to be nearly identical in control and Alzheimer's brains.

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Cloning and sequence analysis of cDNAs encoding human hippocampus N-methyl-d-aspartate receptor subunits: evidence for alternative RNA splicing

Abstract

FEBS. Eett.

Prog. Neurobiol.

Neuron

Trends Pharmacol. Sci.

Biochem. Biophys. Res. Commun.

Neuron

FEBS Lett.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Cloning and sequence analysis of cDNAs encoding human hippocampus $N- methyl- d -aspartate$ receptor subunits: evidence for alternative RNA splicing