(Received for publication, July 27, 1994; and in revised form, September 15, 1994)
From the
The murine N-methyl D-aspartate receptor
subunit NR2C (-3) is encoded by a unique gene composed of 12
translated and three 5`-untranslated exons that spread over
20
kilobases of genomic sequence. The GC-rich promoter that lacks TATA-
and CAAT-positioning elements has two transcriptional start sites
separated by 18 base pairs. One of these sites is located in a
conserved initiator motif and, together with the first four exons,
specifies the 5`-untranslated sequence of 772 nucleotides. In this
sequence, two alternative splice variants were detected that show
identical expression patterns in adult mouse brain. Comparison of
intron positions in genes encoding different members of the glutamate
receptor family confirms a close evolutionary relationship of the NR2C
and NMDAR1 subunit genes.
In the vertebrate central nervous system, ionotropic glutamate
receptors mediate the fast synaptic action of the major excitatory
neurotransmitter, L-glutamate. Traditionally, these receptors
have been categorized into functionally different types:
-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors,
high-affinity kainate receptors, and N-methyl D-aspartate (NMDA) (
)receptors (reviewed in (3, 4, 5, 6, 7, 8) ).
The
-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors
mediate the fast component of excitatory postsynaptic currents, whereas
the slow component is contributed by the NMDA receptors. The NMDA
receptor with its characteristic voltage-dependent Mg
block can be considered as a coincidence detector of pre- and
postsynaptic activity, and its activation is thought to trigger
synaptic modulation underlying memory formation. Excessive glutamate
stimulation can induce neuronal cell death due to the high
Ca
permeability of this channel.
Similar to other
ligand-gated ion channels, the ionotropic glutamate receptors are
composed of subunits. For the NMDA receptor, two types of subunits have
been molecularly characterized, and members of both are needed to form
an active ion channel. The NMDAR1 (NR1) subunit occurs in virtually
every neuron and is likely to be a constituent of all NMDA receptor
subtypes. Four distinct NR2 subunits (NR2A to NR2D for rat; 1 to
4 for mouse) have been
isolated(9, 10, 11, 12) . Their
expression is developmentally controlled and restricted to specific
brain areas(13) . In NR1/NR2 heteromeric channel
configurations, the NR2 subunits determine the subtype-specific channel
characteristics(10, 11, 13, 14, 15) .
The NR1 and NR2 subunits are little related in primary sequence, but
are conserved in their general design. They are expressed as large
precursor polypeptides with an N-terminal signal peptide. The mature
subunits are predicted to contain four hydrophobic transmembrane
sequences (M1 to M4) and a discontinuous agonist-binding
region(14) . The NR2 subunits contain a uniquely large C
terminus of unknown function.
Most members of the ionotropic
glutamate receptor family occur as molecularly different variants
post-transcriptionally modified by alternative splicing and/or RNA
editing. Thus, the NR1 subunit exists in eight different molecular
forms(16, 17) . No splice variants have been reported
for the NR2 subunits. Here we analyze the gene structure of the murine
NR2C (3) subunit with respect to exon-intron organization,
transcriptional start site, alternatively spliced transcripts, and
nucleotide differences between gene and transcript and compare the gene
organization with that of the NR1 subunit.
Figure 1:
The murine NR2C gene and its expression
in adult mouse brain. A, schematic drawing of the NR2C gene
organization. The exon-intron organization is shown in the upper part.
Exons 1-15 are boxed, and shadedboxes represent untranslated exonic areas. Intron 3, which is 6 kb
in length, is not drawn to scale. The two alternative splice variants
between exons 3 and 4, restriction enzyme cleavage sites (S, SalI; H, HindIII; X, XbaI; K, KpnI), and the positions of the translational
start (ATG) and stop (TGA) codons are indicated. The exon alignment of
the NR2C cDNA is depicted in the lower part. Coding areas for the four
putative transmembrane regions (M1 to M4) are shown by filledboxes. Solidlines below the cDNA and
below the genomic DNA indicate the in situ hybridization
oligonucleotides for splice variant 1 (a), splice variant 2 (b), the in vitro RNA antisense probe of the RNase
protection assay (c), and cDNA probes for the Northern (d) and genomic Southern (e) hybridizations. B, in situ hybridization of horizontal mouse (1, 3) and rat (2, 4) brain sections
using specific oligonucleotides for splice variant 1(1, 2) and splice variant 2(3, 4) . C,
analysis of the transcriptional start site by RNase protection. Total
RNA (50 µg; lane1) from BALB/c mouse cerebellum
and Escherichia coli tRNA (50 µg; lane2) were protected with a
P-labeled antisense
RNA generated by in vitro transcription of a 431-bp genomic
fragment derived from the 5`-untranslated region (positions -1142
to -711) and resolved on a 6% polyacrylamide gel. The protected
RNA species that correspond to the transcriptional start sites mapped
by the RACE protocol and cDNA analysis are indicated by arrows. Approximate RNA sizes are in nucleotides. D,
Northern blot analysis depicting the 4.9-kb NR2C mRNA in 10 µg of
poly(A)
RNA of BALB/c mouse cerebellum. E,
Southern blot analysis of 10 µg of mouse liver DNA digested with KpnI (laneK), XbaI (laneX), and HindIII (laneH)
revealing fragments of expected sizes (see A) when hybridized
with a
P-labeled rat cDNA fragment covering exons
12-15.
Recombinant phages carrying the NR2C subunit gene were
isolated from a mouse genomic library using a rat cDNA fragment as a
hybridization probe. Detailed restriction and sequence analysis of the
exonic and most intronic regions in two recombinant overlapping
clones indicated that the coding region of the gene is spread over
12,800 bp. The gene is divided into 12 exons (Fig. 1A),
and all nucleotide sequences flanking the exons (Fig. 2B) match the consensus splice donor and acceptor
sequences. The mouse genomic coding sequence and the reported cDNA
sequence for
3 (10) differ by five nucleotide exchanges.
The nucleotide insertion at position 2924 and the subsequent deletion
at position 3037 might reflect a sequencing error in the mouse cDNA
since the rat cDNA (11, 15) and the mouse genomic
sequences share the same reading frame. Nucleotide substitutions at
positions 2909, 3293 (C to A), and 3407 (C to T) are unique in the
genomic sequences. They affect the amino acid composition of the NR2C
domains and therefore might reflect allelic variations or site-specific
RNA editing (18) in the mouse NR2C locus.
Figure 2: The nucleotide sequences of the transcriptional start site (A), the exon-intron boundaries (B), and the transcriptional stop site (C) of the mouse NR2C gene. Filledcircles in the 5`-untranslated area mark the transcriptional start site (INR) obtained by the RACE protocol. The opencircle indicates the 5`-end of the cloned rat cDNA. Oligonucleotides bs40, bs48, and bs49 used for RACE analysis are indicated by arrows. Blackarrowheads mark the positions of intronic sequences, including the alternative splice acceptor site in exon 4. Sequences that show similarities to nucleotide motifs that interact with transcriptional regulators are boxed (ec, SV40 enhancer core; ELP, embryonal long terminal repeat-binding protein; Sp1, Sp1-binding site; INR, initiator). The translational start site is represented by a bentarrow, the stop codon is indicated by an asterisk, the polyadenylation signal is underlined, and the 3`-terminal nucleotide of the transcript is indicated by an opensquare.
The NR2C gene exists as a single copy in the haploid mouse genome, consistent with the localization of the NR2C gene to human chromosome 7q25(19) . A Southern analysis of murine liver DNA using as a probe a cDNA fragment specific for exons 12-15 revealed a fragment pattern that was consistent with the restriction map generated for isolated NR2C gene fragments (Fig. 1E).
NR2C
mRNA in adult BALB/c mouse brain is 4.9 kb in length (Fig. 1D). This transcript terminates 18 nucleotides 3`
of the polyadenylation signal AUUAAA, 385 bp after the translational
stop (Fig. 2C), as revealed by specific PCR
amplification of the 3`-end of the NR2C mRNA. An additional, minor NR2C
transcript of 12 kb has been observed in a different mouse
strain(10) , but was not detected in BALB/c. The
5`-untranslated leader sequence of the NR2C gene, 772 nucleotides in
length, is interrupted by three introns. This became apparent when the
5`-untranslated sequences of cloned rat NR2C cDNAs were compared with
the mouse genomic sequence. Gene and cDNA sequences diverge upstream of
base -112 and become colinear in three subsequent exons after a
long intron of 6 kb. PCR amplification of the 5`-untranslated region
revealed that two alternative splice products exist for the mouse NR2C
mRNA (Fig. 1A). These differ by 97 nucleotides, and
both forms can be readily detected by in situ hybridization in
the granule cell layer of adult mouse cerebellum (Fig. 1B). In adult rat brain, at least two additional
splice variants exist, one containing an additional 58 nucleotides of
exonic sequence located between exons 3 and 4. The 58-bp exon is
present in one of the rat NR2C cDNA sequences (15) and in
PCR-amplified products of rat brain NR2C cDNA (data not shown), but was
not detected in intron 3 of the mouse gene, as judged by hybridization
analysis. For the transcriptional start site of the NR2C mRNA, two
positions, -772 and -754 nucleotides upstream of the
translational start site, were identified by rapid amplification of
5`-ends (see ``Materials and Methods'') using the antisense
oligonucleotides that prime 42 and 61 nucleotides downstream of the
putative transcriptional start site (Fig. 2A). These
initiation sites were confirmed by RNase protection of radiolabeled
antisense RNA produced from a genomic fragment (positions -1142
to -712) with 50 µg of total RNA from mouse cerebellum. One
prominent RNA fragment corresponding to initiation site -754 was
protected from RNase digestion. The signal for initiation site
-772 was weak and diffuse and was only visible after longer
exposure times. One of the transcriptional start sites (position
-772) is localized in the consensus sequence pattern
YYAT/AYY, which is described as the transcriptional
initiator for polymerase II transcripts in TATA-containing and
TATA-less promoters(20) . As in these initiators, NR2C
transcription starts at the first A within this element.
The region immediately upstream of the identified transcriptional start sites lacks consensus CAAT or TATA sequence motifs, but is highly GC-rich (72-85%), similar to the high GC content of the neighboring, untranslated exon 1. Sequences corresponding to motifs implicated in binding transcription factor Sp1 (GGGGCGGGGC, position -813)(21) , the embryonal long terminal repeat-binding protein ELP (CAAGGTCAC, position -1022)(22) , and the SV40 enhancer core sequence (CTTTCCAC, position -1157) (23) are located 41, 250, and 385 nucleotides, respectively, upstream of the major transcriptional start sites. The biological importance of these sites needs investigating.
The NR2C gene is the first NR2 subunit gene for which a structure has been determined. When compared with the exon-intron organization of other characterized glutamate receptor subunit genes, e.g. rat GluR-B(26) , Caenorhabditis elegans C-C06E1.4(27) , chick kainate-binding protein(28) , and rat NR1(17) , a close evolutionary origin of NR1 and NR2C subunits can be confirmed. Only between the mouse NR2C gene and the rat NR1 gene were most introns found to interrupt the coding area of the genes in similar or even identical positions (introns 4, 11, and 12 of the NR2C gene and introns 2, 16, 17 of the NR1 gene) (Fig. 3). The NR2C gene contains fewer introns than the gene for NR1 since some of the exons encoding the N-terminal NR2C subunit domain and exons encoding M2 and M3 are fused. Thus, in agreement with the phylogenetic tree(24, 25) and the fact that the NR1 subunit forms functional homomeric channels, the NR1 subunit can be considered to be the oldest member of the NMDA receptor subunit family.
Figure 3: Comparison of the NR2C and NR1 genes. The exon-intron organization of the NR2C and NR1 genes is depicted. Exon regions are boxed and numbered. Shaded boxes represent untranslated exonic areas. The putative four transmembrane regions M1 to M4, alternative splice sites, translational initiation codons (ATG), and stop codons (TGA) are indicated. Identical exon-intron boundaries in the NR1 and NR2C genes are marked with blackdots.