Functional expression of a recombinant unitary glutamate receptor from Xenopus, which contains N-methyl-D-aspartate (NMDA) and non-NMDA receptor subunits.

A cDNA encoding a 100-kDa subunit (XenNR1) of the N-methyl-D-aspartate (NMDA) glutamate receptor type has been cloned from Xenopus central nervous system. When XenNR1 is coexpressed in a mammalian cell line with a recently cloned 51-kDa non-NMDA receptor subunit (XenU1), also from Xenopus, it forms a functional unitary receptor exhibiting the pharmacological properties characteristic of both NMDA and non-NMDA receptors. Firstly, XenU1 can replace NR2 subunits, in complementing XenNR1 to introduce the ligand binding properties of a complete NMDA receptor. Second, responses to both NMDA and non-NMDA receptor agonists and antagonists were obtained in patch-clamp recordings from the cotransfected cells, but no significant responses were recorded when the cells were singly transfected. Third, from solubilized cell membranes from the cotransfected cells, an antibody to the NR1 subunit coprecipitated the binding sites of the non-NMDA receptor subunit. The unitary glutamate receptor has a unique set of properties that denote intersubunit interaction, including a glycine requirement for the responses to non-NMDA as well as to NMDA receptor agonists and voltage-dependent block by Mg2+ of the non-NMDA agonist responses.

Human N-methyl-D-aspartate receptor modulatory subunit hNR3: cloning and sequencing of the cDNA and primary structure of the protein.

Several cDNA clones encoding the human N-methyl-D-aspartate receptor modulatory subunit hNR3, were isolated from a human fetal brain library. The hNR3 cDNA demonstrated a 91.3-91.5% nucleotide (nt) identity with the rat NR2B and mouse epsilon 2 cDNAs. The nt sequence of hNR3 would encode a 1484 amino acid (aa) protein that has a 98.4-98.5% identity with the mouse epsilon 2 and rat NR2B subunits.

Human glutamate receptor hGluR3 flip and flop isoforms: cloning and sequencing of the cDNAs and primary structure of the proteins.

Several cDNA clones encoding the human glutamate receptor subunit GluR3 flip and flop isoforms, were isolated from human hippocampus and fetal brain libraries. DNA sequence analysis revealed overlapping clones permitting the reconstruction of full-length GluR3-flip and GluR3-flop cDNAs. The GluR3 cDNAs demonstrated an 94.1-94.7% nucleotide (nt) identity with the corresponding rat cDNAs. The nt sequence of the GluR3 cDNAs would encode 894 amino acid proteins that have a 99.4% identity with the rat GluR3 isoforms. The human GluR3 cDNAs predict an additional 6 amino acid in the N-terminal signal peptide as compared to the rat GluR3.

Cloning and sequence analysis of additional splice variants encoding human N-methyl-D-aspartate receptor (hNR1) subunits.

Two cDNA clones representing previously unidentified human N-methyl-D-aspartate receptor (hNR1) subunit polypeptides were isolated and sequenced. Clone hNR1-4 was isolated from a human hippocampus cDNA library and was presumably generated by alternative RNA splicing in the 3' amino acid (aa) coding regions. The hNR1-4 cDNA demonstrated an 85.7% nucleotide (nt) identity to the corresponding rat NR1 (rNR1) cDNA. The nt sequence of hNR1-4 would encode a protein that has a 99.8% identity with the corresponding rNR1 subunit. Clone hNR1N was isolated by polymerase chain reaction (PCR)-mediated amplification of a 0.6-kb DNA fragment from human cerebellum cDNA. The nt sequence of this DNA fragment was identical to previously isolated hNR1 cDNA clones, except for the presence of a 63-bp DNA insertion that would encode an additional 21 aa. This DNA insertion occurs in the 5' aa coding regions of hNR1 and presumably represents an exon that is subject to alternative splicing. The nt and aa sequences of this exon are identical between human and rat.

Human N-methyl-D-aspartate receptor modulatory subunit hNR2A: cloning and sequencing of the cDNA and primary structure of the protein.

Several cDNA clones encoding the human N-methyl-D-aspartate receptor modulatory subunit hNR2A, were isolated from human hippocampus and fetal brain libraries. DNA sequence analysis revealed overlapping clones permitting the reconstruction of full-length hNR2A cDNA. The hNR2A cDNA demonstrated an 88-89% nucleotide (nt) identity with the corresponding rodent cDNAs. The nt sequence of hNR2A would encode a 1464-aa protein that has a 95.2% identity with the rodent NR2A subunits.

Molecular characterization of the human EAA5 (GluR7) receptor: a high-affinity kainate receptor with novel potential RNA editing sites.

Several cDNA clones encoding EAA5 receptor polypeptides were isolated from a human fetal brain library. The EAA5 cDNAs demonstrated an 88.7-90.1% nucleotide identity with rat GluR7 cDNAs. The nucleotide sequence of EAA5 would encode a 919-amino acid protein, that has a 97.7-98.9% identity with the rat GluR7 receptor. Two variation of the EAA5 cDNA were identified which result in amino acid substitutions in the predicted extracellular amino-terminal region; Ser310-->Ala and Arg352-->Gln. These variations can be attributed to RNA editing involving T-->G and G-->A substitutions. Both the location (with respect to glutamate receptors), and the nucleotides involved, in this putative RNA editing are novel and may therefore involve novel mechanisms. Ligand binding studies with membranes of transfected COS-1 cells expressing EAA5 polypeptides demonstrate a rank order of ligand affinity similar to that observed with the rat GluR7 receptor, and a dissociation constant for kainate (2.72 +/- 0.12 nM (n = 3)) that is approximately 20- to 30-fold higher than that observed for the rat GluR7 receptor. All of the ligands tested had a higher affinity for the human EAA5 receptor as compared to the rat GluR7 receptor. This report provides another example of pharmacological differences for similar receptors across species.

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

Several cDNA clones encoding human N-methyl-D-aspartate receptor (hNR1) 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 (roNR1) subunits. The changes between the predicted aa sequences of hNR1 and the corresponding roNR1 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.

Nuclease hypersensitivity of the rat cytochrome P450IA1 gene.

The bovine pancreatic deoxyribonuclease I (DNAase I) hypersensitivity of the rat cytochrome P450IA1 gene was investigated. A nuclease-hypersensitive region was observed at approximately 3.2 to 5.1 kilobase pairs upstream of exon 1 in adult and fetal rat liver. This region did not necessarily correlate with gene expression following 3-methylcholanthrene induction, although it may determine the potential for inducibility of this gene.

Multiple constitutive but phenobarbital-inducible rat hepatic nuclear RNA species homologous to a novel cytochrome P-450 cDNA.

A cDNA clone, pPB8, representing partial information for a phenobarbital-inducible rat hepatic cytochrome P-450, immunochemically related to cytochrome P-450b and/or P-450e, hybridized to multiple hepatic nuclear RNA species. In addition to the 3.7 +/- 0.2 kb mRNA encoding this novel cytochrome P-450 isozyme, pPB8 hybridized to nuclear RNAs of 4.9 +/- 0.3, 5.4, 5.7 +/- 0.2, and 6.3 +/- 0.1 kb. These nuclear RNAs were constitutively expressed and were inducible to various extents by phenobarbital administration. The time course of induction of these nuclear RNA components suggested product-precursor relationships. A "full-length" cDNA clone, pPB8/7, synthesized from poly(A)+ RNA homologous to pPB8, detected two mRNA species of 4.6 and 1.8 kb. The 4.6 kb nuclear RNA was inducible by 3-methylcholanthrene, Aroclor 1254, and phenobarbital, while the 1.8 kb nuclear RNA was not appreciably affected. It is suggested that pPB8 and pPB8/7 were synthesized from distinct mRNAs that share homology in their 3' regions.

3-Methylcholanthrene-induced expression of the cytochrome P-450c gene.

Transcriptional control of 3-methylcholanthrene-dependent cytochrome P-450c nuclear RNA induction was directly observed in an in vitro rat liver nuclear transcription system. Mercurated and radiolabeled ribonucleotides were incorporated into nuclear RNA transcribed in vitro, which was then isolated using thiopropyl-Sepharose 6B affinity chromatography. Dot hybridization experiments were carried out using bacteriophage M13 subclones of pRSA57 (a cDNA clone for rat serum albumin), pEB339 (a cDNA clone for rat cytochrome P-450c), and clone 46 (a cDNA clone for mouse cytochrome P1-450). The results of these studies demonstrate that 3-methylcholanthrene does not significantly influence the transcription of the rat serum albumin gene, but does increase the transcription of the cytochrome P-450c gene. Nuclear RNA precursors to the cytochrome P-450c mRNA were characterized by Northern blot analysis. Clone 46 hybridized to nuclear RNA species of 6.7 and 4.0 kb, in addition to the 3.0-kb cytochrome P-450c mRNA. pA8 (a genomic clone for rat cytochrome P-450c), hybridized to the same nuclear RNA species in addition to nuclear RNA species of 4.3, 3.4, and 2.2 kb. M13pd15 (a genomic clone containing information for the first intron of the cytochrome P-450c gene) hybridized to nuclear RNA species of 6.7 and 4.3 kb. All of these nuclear RNA species are polyadenylated. The mRNA coding for cytochrome P-450c was induced maximally in hepatic nuclei at 3 h following 3-methylcholanthrene administration. Maximal accumulation of cytochrome P-450c mRNA in hepatic cytosol has been previously shown to occur at approximately 15 h following 3-methylcholanthrene administration (Bresnick, E., Brosseau, M., Levin, W., Reik, L., Ryan, D. E., and Thomas, P. E. (1981) Proc. Natl. Acad. Sci. USA 78, 4083-4087). These data implicate a possible role of nuclear RNA transport in the regulation of induction of cytochrome P-450c, although further investigations are indicated.

Molecular induction by phenobarbital of a rat hepatic form of cytochrome P-450: expression of a 4-kilobase messenger RNA.

A differential screening procedure was employed to isolate a cDNA clone corresponding to a major phenobarbital (PB)-inducible form of rat hepatic cytochrome P-450. The G-C homopolymer-tailing technique was utilized to construct a cDNA library in the PstI site of plasmid pBR322. The library represented PB-induced poly(A+)RNA sequences from hepatic polysomes of 150-g male Sprague-Dawley rats. Hybrid-selection experiments against total PB-inducible RNA were performed with plasmid DNA derived from clones enriched in PB-inducible information. The mRNA molecules that specifically hybridized were subjected to in vitro translation, were immunoprecipitated with antibody raised in rabbits against purified cytochrome P-450b (P. E. Thomas, D. Korzeniowski, D. Ryan, and W. Levin (1979) Arch. Biochem. Biophys. 192, 524-532), and were electrophoresed under sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic conditions. One cDNA clone, designated PB-8, contained a 600-bp insert partially coding for a PB-inducible cytochrome P-450 species that comigrated on SDS-gel electrophoresis with highly purified P-450b. A single injection of PB, 15-18 h before sacrifice, increased the level of polysomal poly(A+)RNA complementary to the isolated cDNA clone by approximately 16-fold. Northern blot hybridizations of polysome-derived poly (A+)RNA, electrophoresed in denaturing agarose gels, demonstrated that the size of the mRNA corresponding to the isolated clone was 4 kb. Isolated heteronuclear RNA species demonstrated a time-dependent increase in the synthesis of a similar 4-kb RNA molecule. By genomic blot hybridization to EcoRI-restricted DNA, at least three complementary DNA fragments migrating at 5.1, 3.2, and 2.9 kb were observed with 32P-labeled PB-8 as a probe. These data, together with restriction endonuclease mapping and partial cDNA sequence information of the PB-8 cDNA, suggest that the PB-8 clone represents a previously unreported cDNA clone for a form of cytochrome P-450 inducible by PB.