Modulatory proteins of human CNS receptors

Abstract

Neurotransmission by excitatory amino acids (EAAs) such as glutamate is mediated via membrane-bound surface receptors. This neurotransmission has been found to be modulated by certain modulatory proteins. DNA coding for a family of such modulatory proteins has now been isolated and the modulatory proteins have been characterized. Herein described are recombinant cell lines which produce these modulatory proteins as heterologous membrane-bound products. Also described are related aspects of the invention, which are of commercial significance, including the use of cell lines which express the modulatory proteins either homomerically, or heteromerically in a complex with an NMDA receptor, as a tool for discovery of compounds which affect the function of the modulatory proteins.

Description

FIELD OF THE INVENTION

[0002] This invention relates to the application of recombinant DNA technology in the field of neurobiology. More particularly, the invention relates to the cloning and expression of DNA coding for proteins which modulate the function of glutamate receptors.

BACKGROUND OF THE INVENTION

[0003] In the mammalian central nervous system (CNS), the transmission of nerve impulses is controlled by the interaction between a neurotransmitter substance released by the “sending” neuron which then binds to a surface receptor on the “receiving” neuron, to cause excitation thereof. L-glutamate is the most abundant neurotransmitter in the CNS, and mediates the major excitatory pathway in vertebrates. Glutamate is therefore referred to as an excitatory amino acid (EAA) and the receptors which respond to it are variously referred to as glutamate receptors, or more commonly as EAA receptors.

[0004] Members of the EAA receptor family can be grouped into three main types based on differential binding to certain glutamate analogs. One type of EAA receptor, which in addition to glutamate also binds the compound NMDA (N-methyl-D-aspartate), is referred to as the NMDA type of EAA receptor. Two other glutamate-binding types of EAA receptor, which do not bind NMDA, are named according to their preference for binding with two other EAA receptor agonists, namely AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate), and kainate (2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetate). Accordingly, receptors which bind glutamate but not NMDA, and which bind with greater affinity to kainate than to AMPA, are referred to as kainate-type EAA receptors. Similarly, those EAA receptors which bind glutamate but not NMDA, and which bind AMPA with greater affinity than kainate are referred to as AMPA-type EAA receptors.

[0005] The glutamate-binding EAA receptor family is of great physiological and medical importance. Glutamateis involved in many aspects of long-term potentiation (learning and memory), in the development of synaptic plasticity, in epileptic seizures, in neuronal damage caused by ischemia following stroke or other hypoxic events, as well as in other forms of neurodegenerative processes. The development of therapeutics which modulate these processes is being slowed by the lack of any homogeneous source of receptor material with which to discover selectively binding drug molecules, which interact specifically at the interface of an appropriate EAA receptor. The brain derived tissues currently used to screen candidate drugs are heterogeneous receptor sources, possessing on their surface many receptor types which interfere with studies of the EAA receptor/ligand interface of interest. The search for human therapeutics is further complicated by the limited availability of brain tissue of human origin. It would therefore be desirable to obtain cells that are genetically engineered to produce only the receptor of interest. With cell lines expressing cloned receptor cDNA, a substrate which is homogeneous for the desired receptor is provided, for drug screening programs.

[0006] Non-human cDNAs which appear to encode the NMDA-type of EAA receptor have recently been identified and isolated. A cDNA encoding a subunit polypeptide of an NMDA receptor in rat, designated NR1, has been isolated as described by Moriyoshi et al. in Nature 354: 31, 1991. An extension of this work has revealed seven isoforms of NR1, presumably generated by combinations of alternative RNA splicing in the amino- and carboxy-terminal regions of NR1 (Anantharam et al. FEBS Lett. 305: 27, 1992; Durand et al. Proc. Natl. Acad. Sci. USA 89: 9359, 1992; Nakanishi et al. Proc. Natl. Acad. Sci. USA 89: 8552, 1992; Sugihara et al. Biochem. Biophys. Res. Commun. 185: 826, 1992; Hollmann et al. Neuron 10: 943, 1993; Kusiak and Norton. Mol. Brain. Res. 20: 64, 1993). DNA encoding NR1 and one of its isoforms have also been cloned from mouse brain by Yamazaki et al. as described in FEBS Lett. 300: 39, 1992. Other rat NMDA receptor subunits, designated NR2A, NR2B, NR2C and NR2D, have also been identified (Monyer et al. Science 256: 1217, 1992; Ishii et al. J. Biol. Chem. 268: 2836, 1993), as well as mouse NMDA receptor subunits which have been designated ε1, ε2, ε3 and ε4 (Meguro et al. Nature 357: 70, 1992; Kutsuwada et al. Nature 358: 36, 1992; Ikeda et al. FEBS Lett. 313: 34, 1992).

[0007] There has emerged from these molecular cloning advances, a better understanding of the structural features of NMDA receptors and their subunits, as they exist in the non-human brain. According to the current model, each NMDA receptor is heteromeric, consisting of individual membrane-anchored subunits, each comprising transmembrane regions and extracellular domains that dictate ligand-binding properties and contribute to the ion-gating function served by the receptor complex.

[0008] In the search for therapeutics useful to treat CNS disorders in humans, it is highly desirable to obtain knowledge of human EAA receptors, and proteins which modulate the activity of these receptors. Such an understanding would provide a means to screen for compounds that selectively interact with this activity, i.e. to stimulate or inhibit receptor activity, thereby providing a means to identify compounds having potential therapeutic utility in humans. Non-human mammalian models are not suitable for this purpose despite significant protein homology due to the fact that minute sequence discrepancies have been found to cause dramatic pharmacological and functional variation between species homologues of the same protein (Oksenberg et al., Nature, 360:161, 1992; Hall et al. Trends Pharmacol. Sci. 14: 376, 1993). It is therefore particularly desirable to provide cloned cDNA encoding human EAA receptor proteins or modulatory proteins thereof, and cell lines expressing these proteins, in order to generate a screening method for compounds therapeutically useful in humans. These, accordingly, are objects of the present invention.

SUMMARY OF THE INVENTION

[0009] Human cDNAs encoding NMDA receptor modulatory proteins have been identified and characterized, and include proteins referred to herein as the NR3 and NR4 modulatory proteins. Specifically encompassed are parent proteins designated the NR3-1 and NR4-1 proteins, as well as functional sequence-related variants of NR3-1 and NR4-1, and functional fragments of NR3-1 and NR4-1.

[0010] In one of its aspects, thus, the present invention provides an isolated polynucleotide, consisting either of DNA or of RNA, which codes for a human NR3 protein, or functional fragments thereof.

[0011] In another aspect of the present invention, there is provided a cell that has been genetically engineered to produce a human EAA receptor modulatory protein belonging to the herein-defined NR3 family. In related aspects of the present invention, there are provided recombinant DNA constructs and methods useful to obtain substantially homogeneous sources of the human NR3 protein, comprising the steps of culturing the genetically engineered cells, and then recovering the cultured cells.

[0012] In another aspect of the present invention, there is provided a method for evaluating interaction between a candidate ligand and a human EAA receptor modulatory protein, which comprises the steps of incubating the candidate ligand with a genetically engineered cell as described above, or with a membrane preparation derived therefrom, and then assessing said interaction by determining the extent of protein/ligand binding, or by determining the ligand-induced electrical current across said cell.

[0013] In yet another aspect of the present invention, a cell that has been engineered genetically to produce a human heteromeric NR3/receptor complex comprising an NR3 protein and an NMDA receptor is provided.

[0014] In a further aspect of the present invention, there is provided a method for evaluating interaction between a candidate ligand and a human heteromeric NR3/receptor complex comprising an NR3 protein and an NMDA receptor, said method comprising the steps of incubating the candidate ligand with a cell line engineered to produce said receptor complex, or with a membrane preparation derived therefrom, and then assessing the interaction therebetween by determining the extent of protein/ligand binding, or by determining the ligand-induced electrical current across said cell.

[0015] Other aspects of the present invention include a human NR3 protein, in a form essentially free from other proteins of human origin, functional and immunogenic fragments of the protein, antibodies which bind to the protein, and oligonucleotides which hybridize to nucleic acid encoding the protein.

[0016] Other aspects of the present invention, which encompass various applications of the discoveries herein described, will become apparent from the following detailed description, and from the accompanying drawings in which:

BRIEF REFERENCE TO THE DRAWINGS

[0017] FIG. 1 provides the nucleotide sequence (SEQ ID NO: 1) of DNA encoding an NR3 modulatory protein, and the deduced amino acid sequence (SEQ ID NO: 2) thereof;

[0018] FIGS. 2A and 2B illustrate, with plasmid maps, the strategy used to construct expression vectors harbouring the DNA sequence illustrated in FIG. 1;

[0019] FIG. 3 provides, with reference to FIG. 1, the partial DNA and amino acid sequences (SEQ ID NOs: 5 & 6) of a naturally occurring variant of the modulatory protein illustrated in FIG. 1;

[0020] FIG. 4 provides the partial nucleotide sequence (SEQ ID NO: 7) of DNA encoding an NR4 modulatory protein, and the deduced amino acid sequence (SEQ ID NO: 8) thereof;

[0021] FIG. 5 provides the nucleotide sequence (SEQ ID NO: 9) of DNA encoding the NR2A-1 probe;

[0022] FIG. 6 provides the nucleotide sequence (SEQ ID NO: 10) of DNA encoding the NMDAR1-1 receptor, and the deduced amino acid sequence (SEQ ID NO: 11) thereof;

[0023] FIG. 7 provides a comparison of partial nucleotide sequences of NMDAR1-1 (SEQ ID NO: 12) with its variants, NMDAR1-2, NMDAR1-3A and NMDAR1-3C (SEQ ID NOs: 13, 14 & 15, respectively);

[0024] FIG. 8 provides a comparison of the amino acid sequences of NMDAR1-1 (SEQ ID NO: 16) and NMDAR1-4 (SEQ ID NO: 17);

[0025] FIG. 9 provides a comparison of the amino acid sequences of NMDAR1-1/2/3/4 (SEQ ID NO: 18) and NMDAR1-5/6/7/8 (SEQ ID NO: 19); and

[0026] FIGS. 10A-10D graphically illustrate electrophysiological properties of a heteromeric complex comprising NR3-1 and NMDAR1-3C.

DETAILED DESCRIPTION OF THE INVENTION AND ITS PREFERRED EMBODIMENTS

[0027] The present invention relates to modulatory proteins of excitatory amino acid (EAA) receptors of human origin, and to isolated polynucleotides encoding them. More particularly, the present invention is directed to novel human modulatory proteins, herein designated the NR3 and NR4 EAA receptor modulatory proteins, which modulate the activity of human EAA receptors of the NMDA-type. The NR3 and NR4 modulatory proteins comprise the NR3-1 and NR4-1 parent proteins, as well as functional sequence-related variants of the human NR3-1 protein and functional fragments of the NR3-1 protein.

[0028] As used herein, the term “modulatory protein” refers to a protein that, when combined with a human EAA receptor, and in particular with a human NMDA receptor, forms a heteromeric receptor complex having electrophysiological properties which are distinct from the electrophysiological properties of a homomeric receptor complex formed from the selected NMDA receptor alone. Thus, the NR3 and NR4 proteins of the present invention have been found to modulate the ion channel activity of NMDA-type receptors, i.e. receptors having a ligand binding profile comprising specific binding affinity for glutamate, NMDA and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate]. The electrophysiological properties, or ion channel activity, of EAA receptors is typically determined using established electrophysiological assays appropriate for detecting conductance across a cell membrane such as the assay described by Hollmann et al. in Nature 342: 643.

[0029] The term “isolated” as it is used herein with respect to NR3-encoding polynucleotides refers to polynucleotides which are free from human DNA which encodes, or partially encodes, CNS proteins other than NR3 proteins and NMDA receptor proteins.

[0030] The term “heteromeric receptor complex” is used to refer to a receptor complex comprising a modulatory protein, in accordance with the present invention, and an NMDA receptor. A “heteromeric NR3/receptor complex” refers to a receptor complex comprising an NR3 modulatory protein and an NMDA receptor.

[0031] Variants of the NR3-1 and NR4-1 parent modulatory proteins also form modulatory proteins as defined above. Specifically included are functional variants which exhibit a modulatory activity similar to that of the parent protein, and which demonstrate substantial sequence homology to the parent protein. Specifically, variants of NR3-1 will share greater than 98.5% amino acid identity with the NR3-1 protein. Variants of the NR3-1 protein include both naturally occurring variants, an example of which is the NR3-2 protein, illustrated in part in FIG. 3 by nucleic acid and amino acid sequence (SEQ ID NOs: 5 & 6), as well as synthetically derived variants of the human NR3-1 protein.

[0032] The term “fragment” is used herein to denote functional segments of an NR3 or NR4 protein.

[0033] Variants and fragments of the NR3 and NR4 proteins are said to be “functional” if, on coexpression with an NMDA receptor in a heteromeric receptor complex as defined above, the complex, when assayed electrophysiologically, exhibits ligand-induced ion channel activity having measurable current, i.e. current which is greater than the current in the absence of the ligand or greater than the “baseline” current, and the channel activity possess properties which are characteristic of an NMDA ion channel, for example the channel activity is blocked by Mg++ ions and by MK-801.

[0034] Each of the naturally occurring members of the human NR3 and NR4 modulatory proteins possess structural features similar to those of EAA receptors, including an extracellular amino-terminal (N-terminal) region, as well as internal hydrophobic domains which serve to anchor the protein within the cell surface membrane. The particular human EAA receptor modulatory protein designated NR3-1 is a protein characterized structurally as a single polypeptide chain that is produced initially in precursor form bearing an N-terminal signal peptide, and is transported to the cell surface in mature form, lacking the signal peptide. The NR3-1 protein, including its signal peptide, consists of 1,484 amino acids arranged in the sequence illustrated, by single letter code, in FIG. 1 (SEQ ID NO: 2). The particular human EAA receptor modulatory protein designated NR4-1 is a protein encoded by the partial nucleotide sequence illustrated in FIG. 4 (SEQ ID NO: 7).

[0035] A naturally occurring structurally-related variant of the NR3-1 protein has also been identified and is designated herein, the NR3-2 modulatory protein. This variant protein differs from its NR3-1 parent by a single amino acid as illustrated in FIG. 3. Specifically, the serine residue at position 407 in NR3-1 is an asparagine residue in the NR3-2 variant. This change is reflected as a single nucleotide difference between the nucleic acid encoding the two proteins, namely a codon change from “AGC” in NR3-1 to “AAC” in NR3-2.

[0036] The NR3 and NR4 proteins are characterized by their modulatory activity particularly with respect to human NMDA-type receptors, and more particularly with respect to NMDA receptors of the NMDAR1 family, which are described in detail in co-pending U.S. patent appln. Ser. No. 07/987,953, the content of which is incorporated herein by reference. The NMDAR1 family of EAA receptors comprises the NMDAR1-1 receptor, the nucleic acid sequence of which is illustrated in FIG. 6 (SEQ ID NO: 10), and variants of the NMDAR1-1 receptor which retain an NMDA-type ligand binding profile and which are structurally related to NMDAR1-1, i.e. share at least 99.6% amino acid identity with the 1-845 amino acid region of the NMDAR1-1 receptor, and preferably share 100% amino acid identity in this region. There are both naturally occurring and synthetically derived variants of the human NMDAR1-1 receptor. Naturally occurring variants include, but are not restricted to, receptor variants designated human NMDAR1-2, NMDAR1-3A and NMDAR1-3C, the partial nucleotide sequences of which are illustrated in FIG. 7 (SEQ ID NOs: 13, 14 & 15, respectively) and compared to the nucleotide sequence of NMDAR1-1 (SEQ ID NO: 12). Another variant, designated NMDAR1-3B, differs in amino acid sequence from the NMDAR1-1 and NMDAR1-3C receptors by a single amino acid at position 470. This amino acid is lysine in NMDAR1-3B and is glutamic acid in NMDAR1-1 and NMDAR1-3C. This change results from a single base pair change in the codon at position 2560 of NMDAR1-1 and NMDAR1-3C from GAG to AAG in the 3B variant. An NMDAR1-4 variant differs from the NMDAR1-1 receptor by a peptide insert between amino acids 845 and 846 of NMDAR1-1 as illustrated in FIG. 8. Further variants include NMDAR1-4, NMDAR1-5, NMDAR1-6, NMDAR1-7 and NMDAR1-8, which correspond respectively to the NMDAR1-1, NMDAR1-2, NMDAR1-3 and NMDAR1-4 receptors additionally including a 21 amino acid insert as illustrated in FIG. 9.

[0037] One of skill in the art will appreciate that variants of any one of the NMDAR1-1 to NMDAR1-8 receptors which include minor variations from the amino acid sequences thereof, e.g. 1 to 6 amino acid substitutions, deletions or additions, and resulting in receptors retaining the ligand binding profile characteristic of NMDA-type receptors, are also encompassed within the NMDAR1 family of receptors.

[0038] Accordingly, the NR3 and NR4 proteins of the present invention are useful in a heteromeric structure to screen for candidate compounds having the ability to alter the activity of the heteromeric receptor complex. In addition, and despite the understanding that the NR3 and NR4 proteins require a heteromeric structure to function in a modulatory sense, cells producing NR3 and NR4 proteins homomerically, independent of association with an NMDA receptor, can be exploited for the purpose of screening candidate ligands for the ability to interact specifically therewith. Those compounds found to interact with an NR3 or an NR4 protein represent potential drug compounds which may have agonist or antagonist properties useful in the treatment of neurological disease conditions.

[0039] For use in assessing interaction between an NR3 or an NR4 protein, either in homomeric or heteromeric form, and a candidate compound, it is desirable to construct by application of genetic engineering techniques a cell that produces a human NR3 or NR4 protein in functional form as a heterologous product. The construction of such cell lines is achieved by introducing into a selected host cell a recombinant DNA construct in which DNA coding for a secretable form of the modulatory protein, i.e. a form bearing either its native signal peptide or a functional, heterologous equivalent thereof, is associated with expression controlling elements that are functional in the selected host to drive expression of the modulatory protein-encoding DNA, and thus elaborate the desired NR3 or NR4 protein. Such cells are herein characterized as having the protein-encoding DNA incorporated “expressibly” therein. The protein-encoding DNA is referred to as “heterologous” with respect to the particular cellular host if such DNA is not naturally found in the particular host.

[0040] It is most desirable to use a mammalian cell host to produce the present modulatory proteins due to their human origin; however, other suitably engineered eukaryotic and prokaryotic hosts may also be employed to produce NR3 proteins. Accordingly, bacterial hosts such as E. coli and B. subtilis, fungal hosts such as Asgergillus and yeast and insect cell hosts such as Spodoptera frugiperda, are examples of non-mammalian hosts that may also be used to produce NR3/NR4 proteins of the present invention.

[0041] The particular cell type selected to serve as host for production of the human modulatory proteins can be any of several cell types currently available in the art. Preferably, where the modulatory protein will be expressed in heteromeric form, i.e. in conjunction with an NMDA receptor, the cell type selected will not be one which in its natural state elaborates a surface receptor that has ion channel activity or that elaborates a protein that is capable of modulating receptor activity, so as to confuse the assay results sought from the engineered cell line. Generally, such problems are avoided by selecting as host a non-neuronal cell type. However, neuronal cells may nevertheless serve as expression hosts, provided that any “background” activity is accounted for in the assay results.

[0042] According to one embodiment of the present invention, the cell line selected to serve as host for modulatory protein production is a mammalian cell. Several types of such cell lines are currently available for genetic engineering work, and these include the chinese hamster ovary (CHO) cells for example of K1 lineage (ATCC CCL 61) including the Pro5 variant (ATCC CRL 1281); fibroblast-like cells derived from SV40-transformed African Green monkey kidney of the CV-1 lineage (ATCC CCL 70), of the COS-1 lineage (ATCC CRL 1650) and of the COS-7 lineage (ATCC CRL 1651); murine L-cells, murine 3T3 cells (ATCC CRL 1658), murine C127 cells, human embryonic kidney cells of the 293 lineage (ATCC CRL 1573), human carcinoma cells including those of the HeLa lineage (ATCC CCL 2), and neuroblastoma cells of the lines IMR-32 (ATCC CCL 127), SK-N-MC (ATCC HTB 10) and SK-N-SH (ATCC HTB 11).

[0043] A variety of gene expression systems have been adapted for use with these hosts and are now commercially available. Any one of these systems can be exploited to drive expression of DNA encoding NR3 or NR4 proteins. These systems, available typically in the form of plasmidic vectors, incorporate expression cassettes, the functional components of which include DNA constituting host-recognizable expression controlling sequences which enable expression of the receptor-encoding DNA when linked 5′ thereof. The systems further incorporate DNA sequences which terminate expression when linked 3′ of the protein-encoding region. Thus, for expression in a selected mammalian cell host, there is generated a recombinant DNA expression construct in which DNA encoding an NR3 or NR4 protein is linked with expression controlling DNA sequences recognized by the host, including a region 5′ of the DNA to drive expression, and a 3′ region to terminate expression. The plasmidic vector harbouring the expression construct typically incorporates such other functional components as an origin of replication, usually virally-derived, to permit replication of the plasmid in the expression host, including bacterial hosts such as E. coli. To provide a marker enabling selection of stably transfected recombinant cells, the vector will also incorporate a gene conferring some survival advantage on the transfectants, such as a gene coding for neomycin resistance in which case the transfectants are plated in medium with neomycin.

[0044] Included among the various recombinant DNA expression systems that can be used to achieve mammalian cell expression of DNA encoding NR3 or NR4 are those that exploit promoters of viruses that infect mammalian cells, such as the promoter from the cytomegalovirus (CMV), the Rous sarcoma virus (RSV), simian virus (SV40), murine mammary tumor virus (MMTV) and others. Also useful to drive expression are promoters such as the long terminal repeat (LTR) of retroviruses, insect cell promoters such as those regulated by temperature, and isolated from Drosophila, as well as mammalian gene promoters such as steroid-inducible promoters and those regulated by heavy metals i.e. the metalothionein gene promoter. In order to achieve expression in bacterial hosts, such as E. coli, expression systems that exploit the expression controlling regions of various E. coli and viral genes can be used to drive NR3/NR4 production including the lac gene, the trp gene, and regions of the lambda genome (PL and PR). Expression in yeast can be achieved using the expression-controlling regions of genes such as alcohol dehydrogenase and melibiase, and in Aspergillus, the expression-controlling regions of genes such as alcohol dehydrogenase and glucoamylase may be used. The expression controlling-regions of baculovirus may be used in the case of insect host cells.

[0045] For incorporation into the recombinant DNA expression vector, DNA coding for the desired modulatory protein, e.g. an NR3 or NR4 protein, can be obtained by applying selected techniques of gene isolation or gene synthesis. As described in more detail in the examples herein, the present modulatory proteins, including naturally occurring variants thereof, are encoded within the human genome, expressed in human brain tissue, and can therefore be obtained by careful application of conventional gene isolation and cloning techniques. This typically will entail extraction of total messenger RNA from a fresh source of human brain tissue, such as cerebellum, hippocampus or fetal brain tissue, followed by conversion of messenger RNA to cDNA and formation of a library in, for example, a bacterial plasmid, or more typically a bacteriophage. Bacteriophage harbouring fragments of the human DNA are typically grown by plating on a lawn of susceptible E. coli bacteria, such that individual phage plaques or colonies can be isolated. The DNA carried by the phage colony is then typically immobilized on a nitrocellulose or nylon-based hybridization membrane, and then hybridized, under carefully controlled conditions, to a radioactively (or otherwise) labelled nucleotide probe of appropriate sequence to identify the particular phage colony carrying NR3 or NR4-encoding DNA of interest. Typically, the gene or a portion thereof so identified is subcloned into a plasmidic vector for nucleic acid sequence analysis.

[0046] Having herein provided the nucleotide sequence of human NR3 modulatory proteins, it will be appreciated that automated techniques of gene synthesis and/or amplification can also be performed to generate DNA coding therefor. Because of the length of NR3-encoding DNA, application of automated synthesis may require staged gene construction, in which regions of the gene up to about 300 nucleotides in length are synthesized individually and then ligated in correct succession for final assembly. Individually synthesized gene regions can be amplified prior to assembly using polymerase chain reaction (PCR) technology as generally described by Barnett et al. in Nucl. Acids Res. 18:3094, 1990.

[0047] The application of automated gene synthesis techniques provides an opportunity to generate sequence variants of naturally occurring members of the NR3 gene family. It will be appreciated, due to the degeneracy associated with nucleotide triplet codons, that variant polynucleotides coding for the NR3 receptors herein described can be generated by substituting synonymous codons for those represented in the naturally occurring polynucleotide sequences herein identified, such as those identified in FIG. 1 and FIG. 3. For example, as would be known by one of skill in the art, arginine may be encoded by any one of six codons selected from CGA, CGC, CGG, CGU, AGA and AGG, threonine may be encoded by any one of four codons selected from ACA, ACC, ACG and ACU, while lysine is encoded by two codons, AAA and AAG. In addition, polynucleotides coding for synthetic variants of the NR3 receptors can be generated which, for example, incorporate one or more, e.g. 1-10, single amino acid substitutions, deletions or additions. Since it will for the most part be desirable to retain the modulatory activity of the NR3 protein for screening purposes, it is desirable to limit amino acid substitutions to those regions which are less critical for modulatory activity as may be elucidated upon domain mapping of the protein. Such substitutions may include, for example, conservative amino acid substitutions such as isoleucine to leucine, or lysine to arginine.

[0048] With appropriate template DNA in hand, the technique of PCR amplification may also be used to directly generate all or part of the final gene. In this case, primers are synthesized which will prime the PCR amplification of the final product, either in one piece, or in several pieces that may be ligated together. This may be via step-wise ligation of blunt-ended, amplified DNA fragments, or preferentially via step-wise ligation of fragments containing naturally occurring restriction endonuclease sites. In this application, it is possible to use either cDNA or genomic DNA as the template for the PCR amplification. In the former case, the cDNA template can be obtained from commercially available or self-constructed cDNA libraries of various human brain tissues, including hippocampus and cerebellum.

[0049] Once obtained, the DNA encoding the desired modulatory protein is incorporated for expression into any suitable expression vector using conventional procedures, and host cells are transfected therewith also using conventional procedures which include, for example, DNA-mediated transformation, electroporation, microinjection, or particle gun transformation. Expression vectors may be selected to provide transfected mammalian cell lines that express the modulatory protein-encoding DNA either transiently or in a stable manner. For transient expression, host cells are typically transfected with an expression vector harbouring an origin of replication functional in a mammalian cell. For stable expression, such replication origins are unnecessary, but the vectors will typically harbour a gene coding for a product that confers on the transfectants a survival advantage, to enable their selection. Genes coding for such selectable markers include, but are not limited to, the E. coli gpt gene which confers resistance to mycophenolic acid, the neo gene from transposon Tn5 which confers resistance to the antibiotic G418 and to neomycin, the dhfr sequence from murine cells or E. coli which changes the phenotype of DHFR(−) cells into DHFR(+) cells, and the tk gene of herpes simplex virus, which makes TK(−) cells phenotypically TK(+) cells. Both transient expression and stable expression can provide transfected cell lines, and membrane preparations derived therefrom, for use in screening assays.

[0050] The recombinant techniques described above can be equally applied to EAA receptor production, in particular NMDA receptor production, as set out in the specific examples described herein and using, for example, the DNA sequences provided in FIGS. 6 and 7, in the preparation of cells which heteromerically produce a modulatory protein and an NMDA receptor. In this case, once the appropriate modulatory protein-encoding and NMDA receptor-encoding expression vectors have been prepared, the cells selected for expression are transfected with a mixture of the expression vectors in the conventional manner.

[0051] For use in screening assays, cells transiently expressing the NR3/NR4-encoding DNA, and the NMDA receptor-encoding DNA, can be stored frozen for later use, but because the rapid rate of plasmid replication will lead ultimately to cell death, usually in a few days, the transfected cells should be used as soon as possible. Such assays may be performed either with intact cells, or with membrane preparations derived from such cells. The membrane preparations typically provide a more convenient substrate for the ligand screening experiments, and are therefore preferred as substrates. To prepare membrane preparations for screening purposes, i.e. ligand binding experiments, frozen intact cells are homogenized while in cold water suspension and a membrane pellet is collected after centrifugation. The pellet is re-suspended and re-centrifuged to remove endogenous ligands that would otherwise compete for binding in the assays. The membranes may then be used as such, or after storage in lyophilized form, in the ligand binding assays. Alternatively, intact, fresh cells harvested about two days after transient transfection or after about the same period following fresh plating of stably transfected cells, can be used for ligand binding assays by the same methods as used for membrane preparations. When cells are used, the cells must be harvested by more gentle centrifugation so as not to damage them, and all washing must be done in a buffered medium, for example in phosphate-buffered saline, to avoid osmotic shock and rupture of the cells.

[0052] The binding of a candidate ligand to a selected human modulatory protein of the invention, or a heteromeric receptor complex comprising a modulatory protein and an NMDA receptor, is evaluated typically using a predetermined amount of cell-derived membrane (measured for example by protein determination), generally from about 25 ug to 100 ug. Competitive binding assays will be useful to evaluate the affinity of a candidate ligand for a heteromeric complex relative to glutamate. This competitive binding assay can be performed by incubating a membrane preparation with radiolabelled glutamate, for example [3H]-glutamate, in the presence of unlabelled candidate ligand added at varying concentrations. Following incubation, either displaced or bound radiolabelled glutamate can be recovered and measured to determine the relative binding affinities of the candidate ligand and glutamate for the particular receptor used as substrate. In this way, the affinities of various compounds for the heteromeric complex can be measured. As will be appreciated by one of skill in the art, binding assays such as radioimmunoassays and ELISA can also be used to determine binding affinity of a candidate ligand. Such competitive binding assays cannot be used in the case of an NR3 or NR4 protein which is expressed homomerically, in a state that does not naturally bind those ligands bound by EAA receptors. Thus, the binding affinity of candidate ligands for the NR3 or NR4 proteins can be determined using a conventional non-competitive type binding assay. Those ligands determined to have an appropriate affinity for the homomeric modulatory protein, i.e. a binding affinity in the micromolar range, and more preferably in the nanomolar range, can then be selected to determine if their binding is specific, and further, if their binding affects the pharmacological and functional characteristics of a heteromeric modulatory protein/receptor complex.

[0053] The NR3 and NR4 proteins of the present invention are functional in a modulatory context, forming heteromeric receptor complexes, comprising a human modulatory protein and an EAA receptor, which exhibit electrophysiological properties that are distinct from those exhibited by either of the modulatory protein or the NMDA receptor components of the complex alone. The modulatory proteins are therefore useful, in the established manner, for screening candidate ligands for their ability to modulate the ion channel activity of such receptor heteromeric complexes. The present invention thus further provides, as a ligand screening technique, a method of detecting interaction between a candidate ligand and a human modultory protein/receptor heteromeric complex, comprising the steps of incubating the candidate ligand with a cell that produces a human modulatory protein/receptor heteromeric complex, or with a membrane preparation derived therefrom, and then measuring the ligand-induced electrical current across said cell or membrane.

[0054] As an alternative to using cells that express the modulatory protein, either homomerically or as a heteromeric receptor complex, ligand characterization may also be performed using cells (for example Xenopus oocytes), that yield functional membrane-bound protein following introduction of messenger RNA coding for the NR3 or NR4 protein, in the case of homomeric expression, or coding for a heteromeric complex, in the case of heteromeric expression. Thus, NR3 or NR4 DNA is typically subcdoned into a plasmidic vector such that the introduced DNA may be easily transcribed into RNA via an adjacent RNA transcription promoter supplied by the plasmidic vector, for example the T3 or T7 bacteriophage promoters. RNA is then transcribed from the inserted gene in vitro, and isolated and purified therefrom for injection into Xenopus oocytes. In the case of a heteromeric complex, the RNA of the NMDA receptor forming the complex is prepared in the same manner for injection into Xenopus oocytes simultaneously with RNA encoding the modulatory protein. Following the injection of nanoliter volumes of an RNA solution, the oocytes are left to incubate for up to several days, and are then tested for the ability to respond to a particular ligand molecule supplied in a bathing solution. In the heteromeric case, due to the fact that an active membrane channel is formed through which ions may selectively pass, the response of a particular ligand molecule in the bathing solution may typically be measured as an electrical current utilizing microelectrodes inserted into the cell or placed on either side of a cell-derived membrane preparation using the “patch-clamp” technique.

[0055] In addition to using DNA encoding the modulatory protein to construct cell lines useful for ligand screening, expression of the DNA can, according to another aspect of the invention, be performed to produce fragments of the protein in soluble form, for structure investigation, to raise antibodies and for other experimental uses. It is therefore desirable in the first instance to facilitate the characterization of particular regions of NR3 or NR4 in quantity and in isolated form, i.e. free from the remainder of the full-length protein. One region of particular interest with regard to the modulatory function of the NR3 and NR4 proteins is the extracellular N-terminal region. To prepare a fragment of the N-terminal region, full-length DNA encoding the modulatory protein may be modified by site-directed mutagenesis, to introduce a translational stop codon into the extracellular N-terminal region, immediately 5′ of the first transmembrane domain (TM1). Since there will no longer be produced any transmembrane domain(s) to “anchor” the protein into the membrane, expression of the modified cDNA will result in the secretion, in soluble form, of only the extracellular N-terminal domain. Standard ligand-binding assays may then be performed to ascertain the degree of binding of a candidate compound to the extracellular domain so produced. Alternatively, a translational stop codon may be introduced downstream of the first transmembrane domain to yield a fragment which retains the ability to anchor into the cell membrane. In this way, a heteromeric channel comprising the N-terminal NR3/NR4 fragment can be formed and used to determine the extent of modulatory activity possessed by the fragment. It may of course be necessary, using site-directed mutagenesis, to produce different versions of this extracellular region, or indeed any other extracellular region of the protein, in order to map the modulatory domain with precision.

[0056] Alternatively, it may be desirable to produce other regions of the modulatory protein, for example all or part of the carboxy-terminus thereof. In this case, site-directed mutagenesis and/or PCR-based amplification techniques may readily be used to provide a defined fragment of the cDNA encoding the domain of interest. Once obtained, such DNA fragments can be expressed in the usual manner, either homomerically to determine if the fragment has ligand-binding activity, or heteromerically to determine the extent to which the fragment retains modulatory activity. Conventional peptide synthetic techniques may also be used to make the desired C-terminal fragments or other fragments, e.g. a desired N-terminal fragment as noted above.

[0057] It will be appreciated that the production of such fragments may be accomplished in a variety of host cells. Mammalian cells such as CHO cells may be used for this purpose, the expression typically being driven by an expression promoter capable of high-level expression, for example, the CMV promoter. Alternately, non-mammalian cells, such as insect Sf9 (Spodoptera frugiperda) cells may be used, with the expression typically being driven by expression promoters of the baculovirus, for example the strong, late polyhedrin protein promoter. Filamentous fungal expression systems may also be used to secrete large quantities of selected domains of the modulatory protein. Aspergillus nidulans for example, with the expression being driven by the alcA promoter, would constitute such an acceptable fungal expression system. In addition to such expression hosts, it will be further appreciated that any prokaryotic or other eukaryotic expression system capable of expressing heterologous genes or gene fragments, whether intracellularly or extracellularly, would be similarly acceptable.

[0058] For use particularly in detecting the presence and/or location of an NR3 or NR4 protein, for example in brain tissue, the present invention also provides, in another of its aspects, antibodies to these proteins. Such antibodies will also have use as diagnostic agents, e.g. to determine if localized amounts or different forms of NR3 or NR4 in selected tissue types are indicative of a disease condition, and as therapeutic agents, by regulating the modulatory activity of an NR3 or NR4 protein on an NMDA receptor ion channel, to prevent disease conditions associated with overactive NMDA receptor ion channels. Preferably, for use therapeutically, the NR3 and NR4 antibodies employed are monoclonal antibodies.

[0059] To raise NR3 antibodies, for example, there may be used as immunogen either the intact, soluble NR3 protein or an immunogenic fragment thereof, produced in a microbial or mammalian cell host as described above or by standard peptide synthesis techniques. Regions of the NR3 protein particularly suitable for use as immunogenic fragments include those corresponding in sequence to an extracellular region of the receptor, or a portion of the extracellular region, such as peptides consisting of residues 27-557, or fragments thereof.

[0060] The raising of antibodies to the desired NR3 or NR4 protein or immunogenic fragment can be achieved, for polyclonal antibody production, using immunization protocols of conventional design, and any of a variety of mammalian hosts, such as sheep, goats and rabbits. For monoclonal antibody production, immunocytes such as splenocytes can be recovered from the immunized animal and fused, using hybridoma technology, to myeloma cells. The fusion cell products, i.e. hybridoma cells, are then screened by culturing in a selection medium, and cells producing the desired antibody are recovered for continuous growth, and antibody recovery. Recovered antibody can then be coupled covalently to a reporter molecule, i.e. a detectable label, such as a radiolabel, enzyme label, luminescent label or the like, optionally using linker technology established for this purpose.

[0061] In detectably labelled form, e.g. radiolabelled form, olignucleotides, including both DNA or RNA, coding for the human NR3 or NR4 modulatory protein and selected regions thereof, may also be used, in accordance with another aspect of the present invention, as hybridization probes for example to identify sequence-related genes resident in the human or other mammalian genomes (or cDNA libraries) or to locate DNA encoding an NR3 or NR4 protein in a specimen, such as brain tissue. This can be done using either the intact coding region, or a fragment thereof, having radiolabelled nucleotides, for example, 32P-labelled nucleotides, incorporated therein. To identify the NR3-encoding DNA in a specimen, it is desirable to use either the full length cDNA coding therefor, or a fragment which is unique thereto. With reference to FIG. 1 and the nucleotide numbering appearing thereon, such nucleotide fragments include those comprising at least about 17 nucleic acids which correspond in sequence to an extracellular region of NR3 DNA, e.g. the N-terminus thereof. Examples of suitable nucleotide fragments are the regions spanning nucleotides 8-1888 and 2732-5976 of NR3-1. Such sequences, and the intact gene itself, may also be used of course to clone NR3-related human genes, particularly cDNA equivalents thereof, by standard hybridization techniques.

[0062] Embodiments of the present invention are described in detail in the following specific Examples which should not be construed as limiting.

EXAMPLE 1

Isolation of DNA Coding for Human NR3-1

[0063] A human NR2A DNA probe corresponding to a portion of nucleotide sequence of NR2A-1, namely the nucleotide region 1832-2361 (SEQ ID NO: 9) as shown in FIG. 5, was generated by PCR-based amplification of recombinant bacteriophage lambda DNA isolated from an Eco RI-based bacteriophage lambda library of human hippocampus cDNA (obtained from Stratagene Cloning Systems, La Jolla, Calif.). The following degenerate oligonucleotide primers were used in the PCR amplification:

   (SEQ ID NO:20)
1) 5′ GGGGTTTAGATCTGGGT-A/C/G/T-ATGATGTT-C/T-GT-A/
   C/G/T-ATG 3′; and
   (SEQ ID NO:21)
2) 5′ GGGGTTTAGATCTGC-A/C/G/T-GC-A/G-TC-A/G-TA-A/
   G/T-AT-A/G-AA-A/G/C/T-GC 3′

[0064] The primers were used at a final concentration of 2 pmol/μl each, in a 50 μl reaction volume (10 mM Tris-HCl, pH 9.0; 50 mM KCl; 1.5 mM MgCl2) containing 100 ng of recombinant human hippocampus cDNA/bacteriophage lambda DNA, 5 units of Thermus aquaticus DNA polymerase (Promega, Madison, Wis.) and 0.2 mM of each deoxyribonucleotide. Thirty-five cycles of amplification proceeded, with denaturation at 95° C. for 1 min., annealing at 50° C. for 1 min., and primer extension at 72° C. for 1 min., followed by a final cycle at 72° C. for 5 min. The 554 bp PCR product was purified from an agarose gel and subcloned into the plasmid vector pT7Blue-T (Novagen, Madison, Wis.) for DNA sequencing.

[0065] A human NR4 DNA probe corresponding to a portion of nucleotide sequence of NR4, namely the nucleotide region 679-1263 as shown in FIG. 4, was also generated as described above.

[0066] The 554 bp human NR2A and NR4 probes were radiolabelled with [α-32P]dCTP using the Amersham Megaprime DNA labelling system (Arlington Heights, Ill.) to a specific activity of 1.0×109 cpm/μg. The labelled probes were used to screen approximately 1×106 plaques of the EcoRI-based human hippocampus cDNA/bacteriophage lambda Zap II library identified above and approximately 800,000 plaques of an Eco RI-based human fetal brain cDNA/bacteriophage lambda Zap II library (obtained from Stratagene). Fifteen positive plaques were identified on replica filters under the following hybridization conditions: 6×SSPE, 50% formamide, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA at 42° C. with 1.85×106 cpm probe per ml hybridization fluid. The filters were washed twice with 2×SSPE, 0.5% SDS at 25° C. for 5 min., followed by a 15 min. wash at 42° C. The filters were exposed to X-ray film (Kodak, Rochester, N.Y.) overnight. The plaques were purified and excised as phagemids according to the supplier's specifications, to generate an insert-carrying Bluescript-SK variant of the phagemid vector.

[0067] DNA sequence analysis of the largest NR3 overlapping clones (isolated as pBS/FB2C and pBS/FB18) revealed a putative ATG initiation codon together with about 210 nucleotides of 5′ untranslated (UTR) information and 4,452 nucleotides of amino acid coding information. This analysis also revealed a termination codon as well as 1,307 nucleotides of 3′ untranslated information. The entire DNA sequence of the NR3-1 cDNA is provided in FIG. 1.

[0068] Partial DNA sequence analysis of the largest NR4 overlapping clones (isolated as pBS/H5A and pBS/H34A) indicated 1,785 nucleotides of amino acid coding information. The DNA sequence of the partial NR4-1 cDNA is provided in FIG. 4.

EXAMPLE 2

Isolation of DNA Coding for the Human NMDAR1-1 Receptor

[0069] A human NMDAR1 probe corresponding to a portion of nucleotide sequence of NMDAR1-1, namely the nucleotide region 2605-3213 as shown in FIG. 6, was generated by PCR-based amplification of recombinant bacteriophage lambda DNA isolated from an Eco RI-based bacteriophage lambda library of human hippocampus cDNA (obtained from Stratagene Cloning Systems, La Jolla, Calif.). The following degenerate oligonucleotide primers were used in the PCR amplification:

   (SEQ ID NO:22)
1) 5′ GGGGTTTGGATCCAA-A/G-GA-A/G-TGGAA-C/T-GGNATGA
   TG 3′; and
   (SEQ ID NO:23)
2) 5′ GGGGTTTAAGCTT-C/T-TC-G/A-TA-G/A-TT-G/A-TG-C/
   T-TT-C/T-TCCAT 3′

[0070] The primers were used at a final concentration of 5 pmol/μl each, in a 50 μl reaction volume (10 mM Tris-HCl, pH 9.0; 50 mM KCl; 1.5 mM MgCl2) containing 100 ng of recombinant human hippocampus cDNA/bacteriophage lambda DNA, 5 units of Thermus aguaticus DNA polymerase (Promega, Madison, Wis.) and 0.2 mM of each deoxyribonucleotide. Thirty-five cycles of amplification proceeded, with denaturation at 94° C. for 1 min., annealing at 51° C. for 1 min., and primer extension at 72° C. for 1 min., followed by a final cycle at 72° C. for 5 min. The 674 bp PCR product was purified from an agarose gel and subcloned into the plasmid vector pTZBlue-T (Novagen, Madison, Wis.) for DNA sequencing.

[0071] The 674 bp human NMDAR1 probe was radiolabelled with [α-32P]dCTP using the Amersham Megaprime DNA labelling system (Arlington Heights, Ill.) to a specific activity of 1.0-2.4×109 cpm/ug. The labelled probe was used to screen approximately 400,000 plaques of an Eco RI-based human hippocampus cDNA/bacteriophage lambda Zap II library. Thirty-five positive plaques were identified on replica filters under the following hybridization conditions: 6×SSC, 50% formamide, 0.5% SDS, 100 ug/ml denatured salmon sperm DNA at 42° C. with 1.85×106 cpm probe per ml hybridization fluid. The filters were washed with 2×SSC, 0.5% SDS at 25° C. for 5 min., followed by 15 min. washes at 37° C. and at 42° C. The filters were exposed to X-ray film (Kodak, Rochester, N.Y.) overnight. Twenty-eight plaques were purified and excised as phagemids according to the supplier's specifications, to generate an insert-carrying Bluescript-SK variant of the phagemid vector.

[0072] DNA sequence analysis of the clone NMDAR1-3C revealed 2,814 nucleotides of amino acid coding information (938 amino acids). The entire DNA sequence of the EcoRI-EcoRI NMDAR1-3C cDNA insert is provided herein by reference to the sequence of NMDAR1-1 set out in FIG. 6 and by reference to the sequence differences between NMDAR1-1 and NMDAR1-3C outlined in FIG. 7. The NMDAR1-3C cDNA was subcloned into the pcDNA1-Amp mammalian expression vector (to form pcDNA1-Amp/hNR1-3C) using standard techniques such as those described below in Example 3 for the subcloning of the NR2A clone into the pcDNA1-Amp vector.

[0073] It will be appreciated that the protocol described above can be used to isolate any of the NMDAR1 receptors in accordance with the present invention.

EXAMPLE 3

Construction of Genetically Engineered Cells Producing a Heteromeric Complex of Human NR3-1 and NMDAR1-3C

[0074] For transient expression in mammalian cells, cDNA coding for human NR3-1 was incorporated into the mammalian expression vector pcDNA1-Amp (Invitrogen Corporation, San Diego, Calif.). This is a multifunctional 5 kbp plasmid vector designed for cDNA expression in eukaryotic systems, and cDNA analysis in prokaryotes. Incorporated on the vector are the CMV immediate early gene promoter and enhancer sequences, SV40 transcription termination and RNA processing signals, SV40 and polyoma virus origins of replication, M13 and ColE1 origins, Sp6 and T7 RNA promoters, and a gene conferring ampicillin resistance. A polylinker is located. appropriately downstream of the CMV and T7 promoters.

[0075] The strategy depicted in FIG. 2 was employed to facilitate incorporation of the NR3-1 cDNA into the expression vector. The FB2C 5′ 5.3 kbp BamHI/Sphl fragment was released from pBS/FB2C and ligated with the 2.5 kbp BamHI/Sphl fragment of pBS/FB18(#20). Restriction-endonuclease digestion was performed to confirm proper insert orientation. The resulting plasmid was termed pBS/hNR3. The 4.8 kbp EcoRI/NotI fragment of pBS/hNR3 was incorporated at the EcoRI/NotI site in the pcDNA1-Amp polylinker. Restriction-endonuclease digestion and DNA sequence analysis was performed to confirm proper insert orientation. The resulting plasmid, designated pcDNA1-Amp/hNR3, was then introduced for transient expression into a selected mammalian cell host, in this case human embryonic kidney cells of the HEK293 lineage (available from the American Type Culture Collection, Rockville, Md.; ATCC CRL 1573).

[0076] The 7.8 kbp plasmid designated pBS/hNR3 carrying the NR3-1 DNA as a 4.8 kbp insert in a 3 kbp pBS plasmid background, was deposited, under the terms of the Budapest Treaty, with the American Type Culture Collection in Rockville, Md., USA on Jun. 3, 1994 under accession number ATCC 75799.

[0077] For transient expression, HEK293 cells were transfected with approximately 0.4 μg DNA (as pcDNA1-Amp/hNR3 or pcDNA1-Amp/hNR1-3C) per 105 HEK293 cells, by lipofectamine-mediated DNA transfection according to the manufacturer's (Life Technologies Inc., Gaithersburg, Md.) specifications. In coexpression experiments, i.e. for heteromeric expression of NR3-1 and NMDAR1-3C, the HEK293 cells were similarly transfected with 0.8 μg of a DNA mixture containing pcDNA1-Amp/hNR3 and pcDNA1-Amp/hNR1-3C. Briefly, HEK293 cells were plated at a density of 105 cells/dish and then grown for 24 hours in 10% FBS-supplemented MEM medium (Life Technologies Inc., Gaithersburg, Md.). The medium was then removed and cells were washed in OPTI-MEM I medium (Life Technologies Inc.) lacking FBS, prior to transfection. A transfection solution (100 μl) containing 2-4 μl of lipofectamine and DNA was then applied to the cells. After incubation for 4 hours at 37° C., cells were washed as previously described and then allowed to grow for 48 hours in 10% FBS-supplemented MEM medium containing 50 μM DL-AP5 (2-amino-5-phosphonopentanoic acid) and 50 μM 7-chlorokyneurinic acid prior to electrophysiological recording.

[0078] In a like manner, stably transfected cell lines can also be prepared using various cell types as host: HEK293, CHO K1 or CHO Pro5. To construct these cell lines, cDNA coding for NR3-1 is incorporated into the mammalian expression vector pRc/CMV (Invitrogen Corp., San Diego, Calif.) which enables stable expression. Insertion of the cDNA places it under the expression control of the CMV promoter and upstream of the polyadenylation site and terminator of the bovine growth hormone gene, and into a vector background comprising the neomycin resistance gene (driven by the SV40 early promoter) as selectable marker. To introduce plasmids constructed as described above, the host cells are first seeded at a density of 5×105 cells/dish in 10% FBS-supplemented MEM medium. After growth for 24 hours, fresh medium is added to the plates and three hours later, the cells are transfected using the lipofectin-mediated DNA transfection procedure according to the manufacturers specifications. Cells resistant to neomycin are selected in 10% FBS-supplemented MEM medium containing G418 (1 mg/ml). Individual colonies of G418-resistant cells are isolated about 2-3 weeks later, clonally selected and then propagated for assay purposes.

EXAMPLE 4

Electrophysiological Characterization

[0079] Standard whole-cell voltage-clamp (Axopatch 1B, Axon Instruments, Foster City, Calif.) techniques were used to record 60 μM NMDA-evoked currents in HEK293 cells transiently transfected as described in Example 3 and expressing hNR3-1 heteromerically with the NMDAR1-3C receptor. The cells were rinsed prior to recording with a solution of 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 10 μM glycine, 5 mM HEPES, pH 7.2 (300 mOsm.). Single electrode, voltage-clamp recordings were carried out using thin-walled borosilicate glass electrodes (WPI-TW150-F4, WPI Inc., Sarasota, Fla.) filled with an intracellular solution of 140 mM CsCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2 (adjusted with 1 M CsOH). NMDA application using a computer controlled array of perfusion barrels allowed for fast application and continuous perfusion with control or 1 mM Mg2+-containing solutions (lag<50 milliseconds).

[0080] The results of the electrophysiological characterization are depicted in FIG. 10. Points at which NMDA was applied are indicated with black bars above the recordings. No NMDA-induced depolarizations were observed in HEK293 cells transiently transfected with NMDAR1-3C alone, or with NR3 alone. NMDA-induced depolarizations were, however, observed in HEK293 cells transiently transfected with both NR3-1 and NMDAR1-3C. As illustrated, these latter currents were blocked by 1 mM MgCl2, a result which is characteristic of NMDA-gated ion channels.

[0081] This electrophysiological characterization indicates that the NR3/NMDA receptor heteromeric complex functions in an authentic manner, and can therefore be used to reliably predict the functional “signature” of its non-recombinant counterpart from intact human brain. These features make the recombinant receptor especially useful for selecting and characterizing ligand compounds which bind to or otherwise modulate the receptor, and/or for selecting and characterizing compounds which may act by displacing other ligands from the receptor. The isolation of the NR3 protein in a pure form, and its expression with an NMDA receptor as a single, homogenous complex, therefore frees the electrophysiological assay from the lack of precision introduced when complex receptor preparations from human and non-human brains are used to attempt such characterizations.

[0082] It will be appreciated that the protocol described above can be used to determine the electrophysiological characteristics of other NR3INMDA heteromeric receptor complexes, such as for example, the NR3-2/NMDAR1-1 complex.

EXAMPLE 5

Ligand-binding Assays on Heteromeric NR3-1/NMDAR1-3C Complex

[0083] Frozen transfected cells, prepared as described in Example 3 above, are resuspended in ice-cold distilled water, sonicated for 5 seconds, and centrifuged for 10 minutes at 50,000×g. The supernatant is discarded and the membrane pellet is stored frozen at −70° C.

[0084] Cell membrane pellets are resuspended in ice cold 50 mM Tris-HCl, pH 7.55, and centrifuged again at 50,000×g for 10 minutes in order to remove endogenous glutamate that would otherwise compete for binding. The pellets are resuspended in ice cold 50 mM Tris-HCl, pH 7.55, and used for the binding experiments described below. Protein concentrations are determined using the Pierce reagent with BSA as an internal standard.

[0085] Binding assays are performed using a 25-100 μg protein equivalent of the cell membrane preparation, and a selected radiolabeled ligand. In particular, for MK-801-binding assays, incubation mixtures consist of 20 nM (+)-[3-3H]MK-801 (30 Ci/mmole), 20 μM glycine, and 1 mM L-glutamate in cold incubation buffer (50 mM Tris-HCl, pH 7.55) at a final volume of 250 μl. Non-specific binding is determined in the presence of 1 mM (+)MK-801. For glutamate binding assays, incubation mixtures consist of 30 nM [3,4-3H]-L-glutamate (47.3 Ci/mmole) in cold incubation buffer at a final volume of 250 μl. Non-specific binding is determined in the presence of 1 mM L-glutamate and displacement is determined in the presence of 1 mM NMDA, 1 mM kainate, or 1 mM AMPA. The reaction mixtures are incubated on ice for 60 minutes in plastic mini-vials. Bound and free ligand are separated by centrifugation for 30 minutes at 50,000×g. The pellets are washed three times in 4 ml of the cold incubation buffer, and then 4 ml of Beckman Ready-Protein Plus scintillation cocktail was added for liquid scintillation counting.

[0086] It will be appreciated that the protocol described above can be used to determine the pharmacological characteristics of other NR3/NMDA heteromeric receptor complexes, such as for example, the NR3-1/NMDAR1-1 complex.

EXAMPLE 6

Ligand-binding Assay for the Homomeric Expression of NR3-1

[0087] Frozen transfected cells, prepared as described in Example 3 above and expressing NR3-1 in the absence of an NMDA receptor, are resuspended in ice-cold distilled water, sonicated for 5 seconds, and centrifuged for 10 minutes at 50,000×g. The supernatant is discarded and the membrane pellet is stored frozen at −70° C.

[0088] Cell membrane pellets are resuspended in ice cold 50 mM Tris-HCl, pH 7.55, and centrifuged again at 50,000×g for 10 minutes in order to remove endogenous ligands that might otherwise compete for binding. The pellets are resuspended in ice cold 50 mM Tris-HCl, pH 7.55, and used for the binding experiments described below. Protein concentrations are determined using the Pierce reagent with BSA as an internal standard.

[0089] Binding assays are performed using a 25-100 μg protein equivalent of the cell membrane preparation, and a selected radiolabeled ligand in cold incubation buffer (50 mM Tris-HCl, pH 7.55) at a final volume of 250 μl. Non-specific binding is determined in the presence of the unlabeled ligand. The reaction mixtures are incubated on ice for 60 minutes in plastic mini-vials. Bound and free ligand are, separated by centrifugation for 30 minutes at 50,000×g. The pellets are washed three times in 4 ml of the cold incubation buffer, and then 4 ml of Beckman Ready-Protein Plus scintillation cocktail are added for liquid scintillation counting.

[0090] Having determined that the selected ligand binds specifically to NR3-1, i.e. that unlabelled ligand competes for binding with the labelled form of that ligand, and that the binding is saturable, the ligand is then tested for its ability to affect the heteromeric expression of NR3-1, i.e. when coexpressed with an NMDA receptor as described above. Appropriate experiments for this purpose include the ligand binding experiment described in Example 5, and the electrophysiological characterization described in Example 4.

EXAMPLE 7

Isolation and Cloning of the NR3-2 Variant

[0091] The procedures described in Examples 1 and 3 for isolating and cloning the NR3-1 protein are applied equally for the isolation and cloning of NR3-2 and other naturally occuring variants of NR3-1, particularly in view of the high sequence homology between the NR3-1 receptor and the NR3-2 variant.

[0092] Moreover, the electrophysiological and ligand-binding assays described in Examples 4, 5 and 6, respectively, are used in the manner described to determine the electrophysiological and ligand binding characteristics of NR3-2 and other NR3-1 variants.

Claims

1. An isolated polynucleotide comprising a region that encodes a human NR3-1 modulatory protein, or a functional fragment thereof which retains the modulatory activity of the NR3-1 protein.

2. An isolated polynucleotide comprising a region encoding a functional variant of a human NR3-1 receptor, wherein said variant shares greater than 98.5% amino acid identity with said NR3-1 protein and retains the modulatory activity of said NR3-1 protein.

3. An isolated polynucleotide as defined in claim 2, comprising a region that encodes a human NR3-2 protein.

4. A recombinant DNA construct having incorporated therein a polynucleotide as defined in claim 1.

5. A recombinant DNA construct having incorporated therein a polynucleotide as defined in claim 2.

6. A cell that has been engineered genetically to produce a human NR3 protein or a fragent thereof, said cell having incorporated expressibly therein a heterologous polynuclotide as defined in claim 1.

7. A cell that has been engineered genetically to produce a human NR3 protein or a fragment thereof, said cell having incorporated expressibly therein a heterologous polynucleotide as defined in claim 2.

8. A membrane preparation derived from a cell as defined in claim 6.

9. A membrane preparation derived from a cell as defined in claim 7.

10. A cell that has been engineered genetically to produce a heteromeric human receptor complex comprising an NR3 protein and an NMDA receptor, said cell having incorporated expressibly therein a heterologous polynucleotide encoding a human NR3 protein and a heterologous polynucleotide encoding a human NMDA receptor.

11. A cell as defined in claim 10 wherein said human NR3 protein is selected from the group consisting of the NR3-1 and the NR3-2 proteins, and the human NMDA receptor is selected from the group consisting of the NMDAR1-1, NMDAR1-2, NMDAR1-3, NMDAR1-4, NMDAR1-5, NMDAR1-6, NMDAR1-7 and NMDAR1-8 receptors.

12. A process for obtaining a substantially homogeneous source of a human NR3 protein selected from the group consisting of the NR3-1 and NR3-2 proteins, said process comprising the steps of culturing cells having incorporated expressibly therein a heterologous polynucleotide encoding NR3-1 or NR3-2, and then recovering the cultured cells.

13. A process for obtaining a substantially homogeneous source of a human NR3 protein according to claim 12, comprising the subsequent step of obtaining a membrane preparation from the cultured cells.

14. A method of assaying a candidate ligand for interaction with a human NR3 protein selected from the group consisting of the NR3-1 and NR3-2 proteins, which comprises the steps of incubating the candidate ligand under appropriate conditions with a cell having incorporated expressibly therein a heterologous polynucleotide encoding NR3-1 or NR3-2, or with a membrane preparation derived therefrom, and then determining the extent of binding between the human NR3 protein and the candidate ligand.

15. A method of assaying a candidate ligand for interaction with a human heteromeric receptor complex comprising an NR3 protein and an NMDA receptor, which comprises the steps of incubating the candidate ligand under appropriate conditions with a cell as defined in claim 10, or with membrane preparation derived therefrom, and then determining the extent of binding between the complex and the candidate ligand, or determining ligand-induced electrical current across said cell or membrane.

16. A human NR3 protein selected from the group consisting of the NR3-1 and the NR3-2 proteins, in a form essentially free from other proteins of human origin.

17. A functional fragment of an NR3 protein selected from the group consisting of the NR3-1 and the NR3-2 proteins.

18. An antibody which binds a human NR3 protein selected from the group consisting of the NR3-1 and the NR3-2 proteins.

19. An immunogenic fragment of a human NR3 protein selected from the group consisting of the NR3-1 and the NR3-2 proteins.

20. An oligonucleotide comprising at least about 17 nucleic acids which hybridizes with a polynucleotide encoding an NR3 protein selected from the group consisting of the NR3-1 and NRe-2 proteins.