Kainate-binding human CNS receptors of the EAA4 family

FIELD OF THE INVENTION

[0002] This invention is concerned with applications of recombinant DNA technology in the field of neurobiology. More particularly, the invention relates to the cloning and expression of DNA coding for excitatory amino acid (EAA) receptors, especially human EAA receptors.

BACKGROUND [TO] 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] Using tissues isolated from mammalian brain, and various synthetic EAA receptor agonists, knowledge of EAA receptor pharmacology has been refined somewhat. Members of the EAA receptor family are now grouped into three main types based on differential binding to such agonists. One type of EAA receptor, which in addition to glutamate also binds the agonist 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. Particularly, 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. Glutamate is 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. However, the development of therapeutics which modulate these processes has been very difficult, due to the lack of any homogeneous source of receptor material with which to discover selectively binding drug molecules, which interact specifically at the interface of the 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 genes, a substrate which is homogeneous for the desired receptor is provided, for drug screening programs.

[0006] Very recently, genes encoding substituent potypeptides of EAA receptors from non-human sources, principally rat, have been discovered. Hollmann et al., Nature 342: 643, 1989 described the isolation from rat of a gene referred to originally as GluR-K1 (but now called simply GluR1). This gene encodes a member of the rat EAA receptor family, and was originally suspected as being of the kainate type. Subsequent studies by Keinanen et al., Science 249: 556, 1990, showed, again in rat, that a gene called GluR-A, which was in fact identical to the previously isolated GluR1, in fact encodes a receptor not of the kainate type, but rather of the AMPA type. These two groups of researchers have since reported as many as five related genes isolated from rat sources. Boulter et al., Science 249: 1033, 1990, revealed that, in addition to GluR1, the rat contained 3 other related genes, which they called GluR2, GluR3, and GluR4, and Bettler et al., Neuron 5: 583, 1990 described GluR5. Keinanen et al., supra, described genes called GluR-A, GluR-B, GluR-C and GluR-D which correspond precisely to GluR1, GluR2, GluR3 and GluR4 respectively. Sommer et al., Science 249: 1580, 1990 also showed, for GluR-A, GluR-B, GluR-C and GluR-D two alternatively spliced forms for each gene. These authors, as well as Monyer et al., Neuron 6: 799, 1991 were able to show that the differently spliced versions of these genes were differentially expressed in the rat brain. In addition to the isolation of these AMPA receptor genes, several studies have more recently attempted to determine the ion-gating properties of different mixtures of the known receptors (Nakanishi et al., Neuron 5: 569, 1990; Hollmann et al., Science 252: 851, 1991; Verdoorn et al., Science 252: 1715, 1991; and see WO 91/06648).

[0007] Some recent work has also been published regarding non-human genes which appear to encode the kainate-type of receptor. Egebjerg et al., Nature 351: 745, 1991, have described the isolation of a gene from rat called GluR6, which although related in sequence to the AMPA receptor genes, forms a receptor which is not activated by AMPA but rather by glutamate, quisqualate, and preferentially, kainate. Other kainate-binding proteins have been described from frog (Wada et al., Nature 342: 684, 1989), chicken (Gregor et al., Nature 342: 689, 1989) and from rat (Werner et al., Nature 351: 742, 1991). These latter genes encode proteins which bind kainate, but which do not readily form into functional ion channels when expressed by themselves.

[0008] There has emerged from these molecular cloning advances a better understanding of the structural features of EAA receptors and their subunits, as they exist in the rat brain. According to the current model of EAA receptor structure, each is heteromeric in structure, consisting of individual membrane-anchored subunits, each having four transmembrane regions, and extracellular domains that dictate ligand binding properties to some extent and contribute to the ion-gating function served by the receptor complex. Keinanen et al, supra, have shown for example that each subunit of the rat GluR receptor, including those designated GluR-A, GluR-B, GluR-C and GluR-D, display cation channel activity gated by glutamate, by AMPA and by kainate, in their unitary state. When expressed in combination however, for example GluR-A in combination with GluR-B, gated ion channels with notably larger currents are produced by the host mammalian cells.

[0009] In the search for therapeutics useful to treat CNS disorders in humans, it is highly desirable of course to provide a screen for candidate compounds that is more representative of the human situation than is possible with the rat receptors isolated to date. It is particularly desirable to provide cloned genes coding for human receptors, and cell lines expressing those genes, in order to generate a proper screen for human therapeutic compounds. These, accordingly, are objects of the present invention.

[0010] It is another object of the present invention to provide in isolated form a DNA molecule which codes for a human EAA receptor.

[0011] It is another object of the present invention to provide a cell that has been genetically engineered to produce a kainate-binding human EAA receptor.

[0012] Other objects of the present invention will be apparent from the following description of the invention.

SUMMARY OF THE INVENTION

[0013] Genes coding for a family of EAA receptors endogenous to human brain have now been identified and characterized. A representative member of this human EAA receptor family, designated human EAA4a, codes for a receptor protein that in addition to binding glutamate with an affinity typical of EAA receptors, also exhibits ligand binding properties characteristic of kainate-type EAA receptors. Sequence-related genes coding for naturally occurring variants of the human EAA4a receptor have also been identified, and constitute additional members of this receptor family, herein referred to as the human EAA4 receptor family.

[0014] The present invention thus provides, in one of its aspects, an isolated polynucleotide, consisting either of DNA or of RNA, which codes for a human EAA4 receptor or for a kainate-binding fragment thereof.

[0015] In another aspect of the present invention, there is provided a cell that has been genetically engineered to produce a kainate-binding, human EAA receptor belonging to the herein-defined EAA4 family. In related aspects of the present invention, there are provided recombinant DNA constructs and relevant methods useful to create such cells.

[0016] In another aspect of the present invention, there is provided a method for evaluating interaction between a test ligand and a human EAA receptor, which comprises the steps of incubating the test ligand with a genetically engineered cell of the present invention, or with a membrane preparation derived therefrom, and then assessing said interaction by determining one of receptor/ligand binding and ligand-mediated ion channel activation.

[0017] 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] DESCRIPTION OF THE DRAWINGS

[0018] FIGS. 1A-1E provide[s] the nucleotide sequence of DNA (SEQ ID NO: 1) coding for an excitatory amino acid receptor of the present invention, and the deduced amino acid sequence thereof (SEQ ID NO: 2);

[0019]
FIG. 2 illustrates with linear plasmid maps the strategy used to construct expression vectors harbouring the DNA sequence illustrated in [FIG. 1] FIGS. 1A-1E;

[0020]
FIG. 3A (SEQ ID NOS: 3-6, respectively and 3B (SEQ ID NOS: 7-8, respectively) show, with reference to [FIG. 1] FIGS. 1A-1E, the DNA and amino acid sequences of naturally occurring variants of the EAA receptor illustrated in [FIG. 1] FIGS. 1A-1E; and

[0021] FIGS. 4-10 illustrate ligand-binding and channel activating properties of the EAA receptor expressed from the coding region provided in [FIG. 1] FIGS. 1A-1E.

DETAILED DESCRIPTION OF THE [INVENTION AND ITS] PREFERRED EMBODIMENTS

[0022] The invention relates to excitatory amino acid (EAA) receptors of human origin, and is directed more particularly to a novel family of kainate-type human EAA receptors, herein designated the human EAA4 receptor family. As used herein, the term “human EAA4 receptor” is intended to embrace the human EAA4a receptor, and kainate-binding variants of the EAA4a receptor that are structurally related thereto, i.e. share at least 98% amino acid identity, including naturally occurring and synthetically derived variants of the EAA4a receptor. Naturally occurring variants of the human EAA4a receptor include particularly the receptors herein designated human EAA4b receptor. Synthetically derived variants of the human EAA4a receptor include kainate-binding variants that incorporate one or more, e.g. 1-10, amino acid substitutions, deletions or additions, relative to the EAA4a receptor.

[0023] As used herein, the term “kainate-binding” refers to receptor variants and receptor fragments that display greater binding affinity for kainate than for either glutamate, AMPA or NMDA, as determined in assays of conventional design, such as the assays herein described.

[0024] Each of the naturally occurring members of the EAA4 receptor family possesses structural features characteristic of the EAA receptors in general, including extracellular N- and C-terminal regions, as well as four internal hydrophobic domains which serve to anchor the receptor within the cell surface membrane. The particular human EAA receptor designated EAA4a is a protein characterized structurally as a single polypeptide chain that is produced initially in precursor form bearing a 31 residue N-terminal signal peptide, and is transported to the cell surface in mature form, lacking the signal peptide and consisting of 877 amino acids arranged in the sequence illustrated, by single letter code, in [FIG. 1] FIGS. 1A-1E. Unless otherwise stated, the term “EAA4 receptor” refers to the mature form of the receptor protein, and amino acid residues of the EAA4 receptors are accordingly numbered with reference to the mature protein sequence. With respect to structural domains of the receptor, hydropathy analysis reveals four putative transmembrane domains, one spanning residues 532-551 inclusive TM-1), another spanning residues 575-593 (TM-2), a third spanning residues 604-622 (TM-3) and the fourth spanning residues 789-809 (TM-4). Based on this assignment, it is likely that the human EAA4a receptor structure, in its natural membrane-bound form, consists of a 531 amino acid N-terminal extracellular domain, followed by a hydrophobic region containing four transmembrane domains and an extracellular, 68 amino acid C-terminal domain.

[0025] As shown in [FIG. 3] FIGS. 3A and 3B, a structurally related variant of the EAA4a receptor, which occurs naturally in human brain tissue, has also been identified and is designated the EAA4b receptor. As deduced from nucleotide sequences of the genes coding for them, the EAA4b variant shares greater than 99% amino acid identity with EAA4a, differing with respect only to a single amino acid change at position 727, which in the EAA4a receptor is a glycine residue and in the EAA4b receptor is an aspartic acid residue.

[0026] In human fetal brain cDNA libraries, the source from which DNA coding for the EAA4a receptor was isolated, the EAA4a receptor is encoded by the nucleotide sequence provided in [FIG. 1] FIGS. 1A-1E. Also isolated during probing of the cDNA library was a polynucleotide variant of the sequence shown in [FIG. 1] FIGS. 1A-1E, designated EAA4a-1, which incorporates a codon different from, but synonymous with, the triplet coding for glutamine at position 621 (see FIG. 3A). It will thus be appreciated that the EAA4a receptor may of course be encoded by polynucleotides incorporating codons synonymous with those illustrated in [FIG. 1] FIGS. 1A-1E.

[0027] Relative to EAA receptors previously discovered in rat tissue, as described in the publications mentioned hereinabove, members of the human EAA4 receptor family share not greater than about 98% amino acid identity with such rat receptors, with the greatest identity of about 97% being shared with the rat GluR6 receptor reported recently by Egebjerg et al., supra. The human EAA4 receptors differ most significantly from this rat receptor in the extracellular, C-terminal region of the receptors which, in the human receptors, is extended by an additional 25 amino acids.

[0028] Like other members of the human EAA4 receptor family, receptor subtype EAA4a is characterized by a pharmacological profile i.e. a ligand binding “signature”, that points strongly to a kainate-type pharmacology, as distinct from other excitatory amino acid receptor types, such as NMDA and AMPA. In addition and despite the understanding that kainate binding receptors require a multi- and perhaps heteromeric subunit structure to function in the pharmacological sense, it has been found that cells producing the unitary EAA4a receptor do, independently of association with other receptor subunits, provide a reliable indication of excitatory amino acid binding and also channel activation. Thus, in a key aspect of the present invention, the human EAA4a receptor is exploited for the purpose of screening candidate compounds for the ability to interact with the present receptors and/or the ability to compete with endogenous EAA receptor ligands and known synthetic analogues thereof, for EAA receptor interaction.

[0029] For use in assessing interaction between the receptor and a test ligand, it is desirable to construct by application of genetic engineering techniques a mammalian cell that produces a human EAA4 receptor 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 human EAA4 receptor, 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 receptor-encoding DNA, and thus elaborate the desired EAA4 receptor protein. Such cells are herein characterized as having the receptor-encoding DNA incorporated “expressibly” therein. The receptor-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.

[0030] The particular cell type selected to serve as host for production of the human EAA4 receptor can be any of several cell types currently available in the art, but should not of course be a cell type that in its natural state elaborates a surface receptor that can bind excitatory amino acids, and so confuse the assay results sought from the engineered cell line. Generally, such problems are avoided by selecting as host a non-neuronal cell type, and can further be avoided using non-human cell lines, as is conventional. It will be appreciated that neuronal- and human-type cells may nevertheless serve as expression hosts, provided that “background” binding to the test ligand is accounted for in the assay results.

[0031] According to one embodiment of the present invention, the cell line selected to serve as host for EAA4 receptor 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); the 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 CAL 1651); murine L-cells, murine 3T3 cells (ATCC CRL 1658), murine Cl 27 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).

[0032] A variety of gene expression systems have been adapted for use with these hosts and are now commercially available, and any one of these systems can be selected to drive expression of the EAA4 receptor-encoding DNA. These systems, available typically in the form of plasmidic vectors, incorporate expression cassettes the functional components of which include DNA constituting expression controlling sequences, which are host-recognized and enable expression of the receptor-encoding DNA when linked 5′ thereof. The systems further incorporate DNA sequences which terminates expression when linked 3′ of the receptor-encoding region. Thus, for expression in the selected mammalian cell host, there is generated a recombinant DNA expression construct in which DNA coding for a secretable form of the receptor is linked with expression controlling DNA sequences recognized by the host, and which include a region 5′ of the receptor-encoding 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 and desirably also for plasmid amplification in a bacterial host, such as E. coli. To provide a marker enabling selection of stably transformed recombinant cells, the vector will also incorporate a gene conferring some survival advantage on the transformants, such as a gene coding for neomycin resistance in which case the transformants are plated in medium supplemented with neomycin.

[0033] Included among the various recombinant DNA expression systems that can be used to achieve mammalian cell expression of the receptor-encoding DNA 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 LTR of retroviruses, insect cell promoters such as those regulated by temperature, and isolated from Drosophila, as well as mammalian gene promoters such as those regulated by heavy metals i.e. the metalothionein gene promoter, and other steroid-inducible promoters.

[0034] For incorporation into the recombinant DNA expression vector, DNA coding for the desired EAA4 receptor, e.g. the EAA4a receptor or a kainate-binding variant thereof, can be obtained by applying selected techniques of gene isolation or gene synthesis. As described in more detail in the examples herein, the EAA4a receptor, and the EAA4b variant thereof, are encoded within the genome of 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 or hippocampus tissue and preferably fetal brain tissue, followed by conversion of message to cDNA and formation of a library in for example a bacterial plasmid, more typically a bacteriophage. Such 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 oligonucleotide probe of appropriate sequence to identify the particular phage colony carrying receptor-encoding DNA or fragment thereof. Typically, the gene or a portion thereof so identified is subcloned into a plasmidic vector for nucleic acid sequence analysis.

[0035] Having herein provided the nucleotide sequence of various human EAA4 receptors, it will be appreciated that automated techniques of gene synthesis and/or amplification can be performed to generate DNA coding therefor. Because of the length of EAA4 receptor-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.

[0036] The application of automated gene synthesis techniques provides an opportunity for generating sequence variants of naturally occurring members of the EAA4 gene family. It will be appreciated, for example and as mentioned above, that polynucleotides coding for the EAA4 receptors herein described can be generated by substituting synonymous codons for those represented in the naturally occurring polynucleotide sequences herein identified. In addition, polynucleotides coding for synthetic variants of the EAA4 receptors herein described can be generated which for example incorporate one or more single amino acid substitutions, deletions or additions. Since it will for the most part be desirable to retain the natural ligand binding pro file of the receptor for screening purposes, it is desirable to limit amino acid substitutions, for example to the so-called conservative replacements in which amino acids of like charge are substituted, and to limit substitutions to those sites less critical for receptor activity e.g. within about the first 20 N-terminal residues of the mature receptor, and such other regions as are elucidated upon recaptor domain mapping.

[0037] 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.

[0038] Once obtained, the receptor-encoding DNA is incorporated for expression into any suitable expression vector, and host cells are transfected therewith using conventional procedures, such as DNA-mediated transformation, electroporation, microinjection, or particle gun transformation. Expression vectors may be selected to provide transformed cell lines that express the receptor-encoding DNA either transiently or in a stable manner. For transient expression, host cells are typically transformed 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 transformants a survival advantage, to enable their selection. Genes coding for such selectable markers include 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 transformed cell lines, and membrane preparations derived therefrom, for use in ligand screening assays.

[0039] For use in screening assays, cells transiently expressing the 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 transformed 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 binding experiments, and are therefore preferred as binding 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 then washed in cold water, and dialyzed to remove endogenous EAA ligands such as glutamate, that would otherwise compete for binding in the assays. The dialyzed 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.

[0040] The binding of a candidate ligand to a selected human EAA4 receptor of the invention 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. Generally, competitive binding assays will be useful to evaluate the affinity of a test compound relative to kainate. This competitive binding assay can be performed by incubating the membrane preparation with radiolabelled kainate, for example [3H]-kainate, in the presence of unlabelled test compound added at varying concentrations. Following incubation, either displaced or bound radiolabelled kainate can be recovered and measured, to determine the relative binding affinities of the test compound and kainate for the particular receptor used as substrate. In this way, the affinities of various compounds for the kainate-type human EAA receptors can be measured.

[0041] The EAA4 receptors of the present invention are per se functional in an electrophysiological context, and are therefore useful, in the established manner, in screening test ligands for their ability to modulate ion channel activity. The present invention thus further provides, as a ligand screening technique, the method of detecting interaction between a test ligand and a human CNS receptor, which comprises the steps of incubating the test ligand with a human EAA4 receptor-producing cell or with a membrane preparation derived therefrom, and then measuring ligand-induced electrical current across said cell or membrane.

[0042] As an alternative to using cells that express receptor-encoding DNA, ligand characterization, either through binding or through ion channel formation, may also be performed using cells for example Xenopus oocytes, that yield functional membrane-bound receptor following introduction of messenger RNA coding for the EAA4 receptor. In this case, the EAA4 receptor gene of the invention is typically subcloned into a plasmidic vector such that the introduced gene 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. ANA is then transcribed from the inserted gene in vitro, and can then be injected into Xenopus oocytes. Following the injection of nL 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. Since functional EAA receptors act in part by operating a membrane channel through which ions may selectively pass, the functioning of the receptor in response to 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 so-called “patch-clamp” technique.

[0043] In addition to using the receptor-encoding DNA 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 receptor in soluble form, for structure investigation, to raise antibodies and for other experimental uses. It is expected that the portion of the EAA4 receptor responsible for binding a ligand molecule resides on the outside of the cell, i.e., is extracellular. It is therefore desirable in the first instance to facilitate the characterization of the receptor-ligand interaction by providing this extracellular ligand-binding domain in quantity and in isolated form, i.e., free from the remainder of the receptor. To accomplish this, the full-length EAA4 receptor-encoding DNA may be modified by site-directed mutagenesis, so as to introduce a translational stop codon into the extracellular N-terminal region, immediately before the sequence encoding the first transmembrane domain (TM1), i.e., before residue 532 as shown in [FIG. 1] FIGS. 1A-1E. Since there will no longer be produced any transmembrane domain(s) to “anchor” the receptor into the membrane, expression of the modified gene will result in the secretion, in soluble form, of only the extracellular ligand-binding 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. It may of course be necessary, using site-directed mutagenesis, to produce several different versions of the extracellular regions, in order to optimize the degree of ligand binding to the isolated domains.

[0044] Alternatively, it may be desirable to produce an extracellular domain of the receptor which is not derived from the amino-terminus of the mature protein, but rather from the carboxy-terminus instead, for example domains immediately following the fourth transmembrane domain (TM4), i.e., residing between amino acid residues 810 and 877 inclusive of FIG. 1. In this case, site-directed mutagenesis and/or PCR-based amplification techniques may readily be used to provide a defined fragment of the gene encoding the receptor domain of interest.

[0045] Such a DNA sequence may be used to direct the expression of the desired receptor fragment, either intracellularly, or in secreted fashion, provided that the DNA encoding the gene fragment is inserted adjacent to a translation start codon provided by the expression vector, and that the required translation reading frame is carefully conserved.

[0046] It will be appreciated that the production of such extracellular ligand binding domains 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 (cytomegalovirus) 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 such extracellular domains of the EAA receptor. Aspergillus nidulans, for example, with the expression being driven by the alcA promoter, would constitute such an acceptable 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.

[0047] For use particularly in detecting the presence and/or location of an EAA4 receptor, for example in brain tissue, the present invention also provides, in another of its aspects, labelled antibody to a human EAA4 receptor. To raise such antibodies, there may be used as immunogen either the intact, soluble receptor 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 EAA4a receptor 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 1-531, including particularly residues 184-199 or 484-527, and peptides corresponding to the region between transmembrane domains TM-2 and TM-3, such as a peptide consisting of residues 594-603. Peptides consisting of the C-terminal domain (residues 810-877), or fragment thereof such as a peptide consisting of residues 850-877 may also be used for the raising of antibodies. Substantially the same region of the human EAA4b receptor may also be used for production of antibodies against this receptor.

[0048] The raising of antibodies to the desired EAA4 receptor 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. Alternatively, for monoclonal antibody production, immunocytes such as splenocytes can be recovered from the immunized animal and fused, using hybridoma technology, to a myeloma cells. The fusion products are then screened by culturing in a selection medium, and cells producing antibody are recovered for continuous growth, and antibody recovery. Recovered antibody can then be coupled covalently to a detectable label, such as a radiolabel, enzyme label, luminescent label or the like, using linker technology established for this purpose.

[0049] In detectably labelled form, e.g. radiolabelled form, DNA or RNA coding for the human EAA4 receptor, 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 the EAA4-encoding DNA in a specimen, such as brain tissue. This can be done using either the intact coding region, or a fragment thereof having radiolabelled e.g. 32P, nucleotides incorporated therein. To identify the EAA4-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] FIGS. 1A-1E and the nucleotide numbering appearing thereon, such nucleotide fragments include those comprising at least about 17 nucleic acids, and otherwise corresponding in sequence to a region coding for the N-terminus or C-terminus of the receptor, or representing a 5′-untranslated or 3′-untranslated region thereof, such as one of the following nucleotide regions: 58-77, 148-165, 347-364, 806-823, 1986-2004, 2395-2413 and 2756-2773 [(FIG. 1)] (FIGS. 1A-1E). These sequences, and the intact gene itself, may also be used of course to clone EAA4-related human genes, particularly cDNA equivalents thereof, by standard hybridization techniques.

EXAMPLE 1

Isolation of DNA Coding for the Human EAA4a Receptor

[0050] cDNA coding for the human EAA4a receptor was identified by probing human fetal brain cDNA that was obtained as an EcoRI-based lambda phage library (lambda ZAP) from Stratagene Cloning Systems (La Jolla, Calif., U.S.A.). The cDNA library was screened using an oligonucleotide probe capable of annealing to the 3′ region of the rat GluR5 receptor sequence reported by Bettler et al., supra. The specific sequence of the 32P-labelled probe (SEQ ID NO: 9) is provided below:

[0051] 5′-ATCGGCGGCATCTTCATTGTTCTGGCTGCAGGACTCGTGC-3′

[0052] The fetal brain cDNA library was screened under the following hybridization conditions; 6× SSC, 25% formamide, 5% Denhardt's solution, 0.5% SOS, 100 ug/ml denatured salmon sperm DNA, 42C. Filters were washed with 2× SSC containing 0.5% SDS at 25C. for 5 minutes, followed by a 15 minute wash at 50C. with 2× SSC containing 0.5% SDS. The final wash was with 1× SSC containing 0.5% SDS at 50C for 15 minutes. Filters were exposed to X-ray film (Kodak) overnight. Of 106 clones screened, only one cDNA insert, of about 2.9 kb, was identified, and designated RKCS5F94. For sequencing, the '94 phage was plaque purified, then excised as a phagemid according to the supplier's specifications, to generate an insert-carrying Bluescript-SK variant of the phagemid vector. Sequencing of the '94 clone across its entire sequence revealed a putative ATG initiation codon together with about 133 bases of 5′non-coding region and 2,724 bases of coding region. Also revealed was a termination codon, as well as about 18 bases of 3′ non-translated sequence. The entire sequence of the EcoRI/EcoRI insert is provided in [FIG. 1] FIGS. 1A-1E.

[0053] A 5.9 kb phagemid designated pBS/humEAA4a, carrying the receptor-encoding DNA as a 2.9 kb EcoRI/EcoRI insert in a 3.0 kb Bluescript-SK phagemid background, was deposited, under the terms of the Budapest Treaty, with the American Type Culture Collection in Rockville, Md. USA on May 28, 1992, and has been assigned accession number ATCC 75245.

EXAMPLE 2

Construction of Genetically Engineered Cells Producing the Human EAA4a Receptor

[0054] For transient expression in mammalian cells, cDNA coding for the human EAA4a receptor was incorporated into the mammalian expression vector pcDNA1, which is available commercially from Invitrogen Corporation (San Diego, Calif., USA; catalogue number V490-20). This is a multifunctional 4.2 kb plasmid vector designed for cDNA expression in eukaryotic systems, and cDNA analysis in prokaryotes. Incorporated on the vector are the CMV promoter and enhancer, splice segment and polyadenylation signal, an SV40 and Polyoma virus origin of replication, and M13 origin to rescue single strand DNA for sequencing and mutagenesis, Sp6 and T7 RNA promoters for the production of sense and anti-sense RNA transcripts and a Col E1-like high copy plasmid origin. A polylinker is located appropriately downstream of the CMV promoter (and 3′ of the T7 promoter).

[0055] The strategy depicted in FIG. 2 was employed to facilitate incorporation of the EAA4a receptor-encoding cDNA into an expression vector. The cDNA insert was released from pBS/humEAA4a as a 2.9 kb PstI/XhoI fragment, which was then incorporated at the PstI/XhoI sites in the pcDNA1 polylinker. Sequencing across the junctions was performed, to confirm proper insert orientation in pcDNA1. The resulting plasmid, designated pcDNA1/humEAA4a, was then introduced for transient expression into a selected mammalian cell host, in this case the monkey-derived, fibroblast like cells of the COS-1 lineage (available from the American Type Culture Collection, Rockville, Md. as ATCC CRL 1650).

[0056] For transient expression of the EAA4a-encoding DNA, COS-1 cells were transfected with approximately 8 ug DNA (as pcDNA1/humEAA4a) per 106 COS cells, by DEAE-mediated DNA transfection and treated with chloroquine according to the procedures described by Maniatis et al., supra. Briefly, COS-1 cells were plated at a density of 5×106 cells/dish and then grown for 24 hours in FBS-supplemented DMEM/F12 medium. Medium was then removed and cells were washed in PBS and then in medium. There was then applied on the cells 10 ml of a transfection solution containing DEAE dextran (0.4 mg/ml), 100 uM chloroquine, 10% NuSerum, DNA (0.4 mg/ml) in DMEM/F12 medium. After incubation for 3 hours at 37C., cells were washed in PBS and medium as just described and then shocked for 1 minute with 10% DMSO in DMEM/F12 medium. Cells were allowed to grow for 2-3 days in 10% FBS-supplemented medium, and at the end of incubation dishes were placed on ice, washed with ice cold PBS and then removed by scraping. Cells were then harvested by centrifugation at 1000 rpm for 10 minutes and the cellular pellet was frozen in liquid nitrogen, for subsequent use in ligand binding assays. Northern blot analysis of a thawed aliquot of frozen cells confirmed expression of receptor-encoding cDNA in cells under storage.

[0057] In a like manner, stably transfected cell lines can also be prepared using two different cell types as host: CHO K1 and CHO Pro5. To construct these cell lines, cDNA coding for human EAA4a is incorporated into the mammalian expression vector pRC/CMV (Invitrogen), which enables stable expression. Insertion at this site placed the cDNA under the expression control of the cytomegalovirus 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.

[0058] To introduce plasmids constructed as described above, the host CHO cells are first seeded at a density of 5×105% 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 calcium phosphate-DNA co-precipitation procedure (Maniatis et al., supra). Briefly, 3 ug of DNA is mixed and incubated with buffered calcium solution for 10 minutes at room temperature. An equal volume of buffered phosphate solution is added and the suspension is incubated for 15 minutes at room temperature. Next, the incubated suspension is applied to the cells for 4 hours, removed and cells were shocked with medium containing 15% glycerol. Three minutes later, cells are washed with medium and incubated for 24 hours at normal growth conditions. Cells resistant to neomycin are selected in 10% FBS-supplemented alpha-MEM medium containing G418 (1 mg/ml). Individual colonies of G418-resistant cells are isolated about 2-3 weeks later, clonally selected and then propogated for assay purposes.

EXAMPLE 3

Ligand Binding Assays

[0059] Transfected cells in the frozen state were resuspended in ice-cold distilled water using a hand homogenizer and centrifuged for 20 minutes at 50,000 g. The supernatant was discarded and the membrane pellet stored frozen at −70° C.

[0060] COS cell membrane pellets were suspended in ice cold 50 mM Tris-HCl (pH 7.55, 5° C.) and centrifuged again at 50,000 g for 10 minutes in order to remove endogenous glutamate that would compete for binding. Pellets were resuspended in ice cold 50 mM Tris-HCl (pH 7.55) buffer and the resultant membrane preparation was used as tissue source for binding experiments described below. Proteins were determined using the Pierce Reagent with BSA as standard.

[0061] Binding assays were then performed, using an amount of COS-derived membrane equivalent to 25-100 ug as judged by protein determination and selected radiolabelled ligand. In particular, for kainate binding assays, incubation mixtures consisted of 25-100 pg tissue protein and [vinylidene-3H1 kainic acid (58 Cl/mmole, B5 nM final) in the cold incubation buffer, 1 ml final volume. Non-specific binding was in the presence of 1 mM L-glutamate. Samples were incubated on ice for 60 minutes, and bound and free ligand were then separated by rapid filtration using a PHD cell harvester and GF/B filters pre-soaked in ice-cold 0.3% polyethyleneimine. Filters were washed twice in 4 ml of the cold incubation buffer, then placed in scintillation vials with 5 ml of Beckman Ready-Protein Plus scintillation cocktail for counting.

[0062] For AMPA-binding assays, incubation mixtures consisted of 25-100 ug tissue protein and D, L-alpha-[5-methyl-3H]amino-3-hydroxy-5-methylisoxazote-4-propionic acid (3H-AMPA, 27.6 Ci/mmole, 10 nM final) with 0.1 M KSCN and 2.5 mM CaCl2 in the 1 ml final volume. Non-specific binding was determined in the presence of 1 mM L-glutamate. Samples were incubated on ice for 60 minutes in plastic minivials, and bound and free ligand were separated by centrifugation for 30 minutes at 50,000 g; Pellets were washed twice in 4 ml of the cold incubation buffer, then 5 ml of Beckman Ready-Protein Plus scintillation cocktail was added, for counting.

[0063] Assays performed in this manner, using membrane preparations derived from the EAA4a-producing COS cells, revealed specific [3H]-kainate binding at 85 nM, labelled ligand (FIG. 4). Mock transfected cells exhibited no specific binding of any of the ligands tested. These results demonstrate clearly that the human EAA4a receptor is binding kainate specifically. This activity, coupled with the fact that there is little or no demonstrable binding of either AMPA or NMDA clearly assigns the EAA4a receptor to be of the kainate type of EAA receptor. Furthermore, this binding profile indicates that the receptor is functioning in an authentic manner, and can therefore reliably predict the ligand binding “signature” of its non-recombinant counterpart from the intact human brain. These features make the recombinant receptor especially useful for selecting and characterizing ligand compounds which bind to the receptor, and/or for selecting and characterizing compounds which may act by displacing other ligands from the receptor. The isolation of the EAA4a receptor gene in a pure form, capable of being expressed as a single, homogenous receptor species, therefore frees the ligand binding assay from the lack of precision introduced when complex, heterogeneous receptor preparations from human brains are used to attempt such characterizations.

EXAMPLE 4

Channel Activity Assays

[0064]

Xenopus oocytes
(stage V or VI) were harvested and the nucleus of the oocytes was injected with 5 ng of pcDNAI/humEAA4a DNA. The oocytes were then tested each day using a two-electrode voltage clamp, according to the method reported by Verdoorn et al, Mol. Pharmacol., 1988, 34:298. Successful expression occurred from day 5 onward.

[0065] Oocytes injected with pcDNAI/humEAA4a and held at −100 mV responded to kainate. The application of kainate at concentrations greater than 5×10−8 M evoked large inward currents (>200 nA) that rapidly desensitized in the continuing presence of agonist (FIG. 5). Full recovery from desensitization caused by 60 second application of kainate required approximately 10 minutes, although at high concentrations (>10−4M), full recovery from desensitization required 15-20 minutes.

[0066] Because of the very large currents induced by the expression of humEAA4a, a dose-response curve for kainate was constructed at the unusually low holding membrane potential of −30 mV (FIG. 6). The concentration of kainate needed to evoke 50% of the maximal response (EC50) was about 5×10−8M which is considerably lower than the EC5, of 10−6M reported by Egejberg et al, supra, for the kainate-binding rat receptor, GluR6.

[0067] The current/voltage (I/V) relationship (FIG. 7) was constructed using 5×10−8M kainate. The I/V relationship was non-linear and exhibiting a strong rectification. It was not possible to reverse the kainate induced currents, although the current disappeared at −10 mV. Furthermore, the currents recorded in oocytes after day 6 were always larger than 2000 nA at membrane potentials greater than −80 mV.

[0068] Exposure of the oocytes to L-glutamate induced smaller currents than kainate, which also rapidly desensitized in the continuing presence of glutamate (FIG. 8). Full recovery from desensitization following 60 second exposure to glutamate required approximately 5 minutes at concentrations below 10−4M and approximately 10 minutes at concentrations above 10−4M.

[0069] Because currents evoked by L-glutamate were smaller than those evoked by kainate, it was possible to construct a dose-response curve at a holding potential of −100mV (FIG. 10). The concentration of L-glutamate required to evoke 50% of the maximal response (EC50) was 5×10−6M, as compared with 3.1×10−5M reported for the rat GluR6 receptor (Egebjerg et al, supra).

[0070] DNQX (6,7-dinitroquinoxaline-2,3-dione) which is an antagonist of non-NMDA receptors blocked the glutamate response (FIG. 8) in a competitive manner. At a concentration of 2×10−6M, DNQX caused a parallel shift of the dose/response curve for L-glutamate to the right (FIG. 10).

[0071] A current-voltage relationship was also constructed for 10−4M L-glutamate. The I/V relationship for L-glutamate (as for kainate) was strongly rectified (FIG. 9).

[0072] Oocytes did not respond to AMPA (10−4M), NMDA (10−4M), or NMDA (10−4M) with glycine (10−6M), when tested at −60, −80 and −100 mV.

[0073] The electrophysiological properties observed in oocytes following injection of pcDNAI/humEAA4a DNA into the nucleus, as well as the observed ligand binding pharmacological profile, indicate that humEAA4a receptor is per se sufficient, in the absence of other receptor complex components that may exist naturally, to form an active receptor/ion channel complex.

[0074] These electrophysiological and pharmacological properties of human EAA4 receptors indicate that the receptor ion-channel complex is functioning in an authentic manner and can therefore reliably predict the electrophysiological properties and ligand binding signature of its non-recombinant counterpart from the intact human brain. These features make the recombinant receptor especially useful for selecting and characterizing functional ligand compounds which can modulate, or effect, an ion channel response of these receptors. Additionally, these receptor/ion channel complexes can be used to identify and characterize test ligands that can block the ion channel function. The isolation of the human EAA4 receptor gene ma pure form, capable of being expressed as a single, homogeneous receptor/ion channel complex, therefore frees the functional ligand assay from the lack of precision introduced when complex, heterogeneous receptor preparations from human brain, and from model mammalian systems such as rat, are used to attempt such characterizations.

	Number	Date	Country
Parent	08249241	May 1994	US
Child	10126617	Apr 2002	US
Parent	07903456	Jun 1992	US
Child	10126617	Apr 2002	US

Kainate-binding human CNS receptors of the EAA4 family

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