ES2372808T3 - HUMAN GLUTAMATE METABOTROPIC RECEPTOR SUBTIPOS (HMR6) AND RELATED DNA COMPOUNDS. - Google Patents

Abstract

A purified human metabotropic glutamate (hmGluR) receptor comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO: 16.

Description

Subtypes of the human glutamate metabotropic receptor (HMR6) and related DNA compounds

The present invention relates to human glutamate metabotropic receptor proteins (hmGluR), with isolated nucleic acids encoding them, with the host cells that produce the proteins of the invention, with the methods for preparing such proteins, with nucleic acids. and with host cells, and with the uses thereof. In addition, the invention provides antibodies directed against the hmGluR proteins of the invention.

Metabotropic glutamate receptors (hmGluR) belong to the class of G-protein-coupled receptors (protein that binds to the guanine nucleotide) that after binding of a glutamatergic ligand can transduce an extracellular signal through a second intracellular messenger system such as Calcium ions, a cyclic nucleotide, diacylglycerol and inositol 1,4,5-triphosphate in a physiological response. Possessing seven putative segments that extend transmembrane, preceded by a large extracellular domain in the amino terminal and followed by a large domain in the carboxyl terminal, metabotropic glutamate receptors are characterized by a common structure. Based on the degree of sequence identity at the amino acid level, the mGluR class can be divided into different subfamilies comprising individual receptor subtypes (Nakanishi, Science 258, 597-603 (1992)). Each subtype of mGluR is encoded by a unique gene. In relation to the homology of an individual subtype of mGluR with another subtype of a different subfamily, the amino acid sequences are approximately less than 50% identical. Within a subfamily the degree of sequence identity is generally approximately less than 70%. Thus, a particular subtype can be characterized by its amino acid sequence homology with another subtype of mGluR, especially a subtype of the same mammalian species. In addition, a particular subtype can be characterized by its tissue and region distribution, its cellular and subcellular expression pattern or by its unmistakable physiological profile, for example by its electrophysiological and pharmacological properties.

As the amino acid L-glutamate is the main excitatory neurotransmitter, it is presumed that glutamatergic systems play an important role in numerous neuronal processes including rapid synaptic excitatory transmission, regulation of neurotransmitter releases, long-term potentiation, learning and memory, plasticity in synaptic development, ischemic hypoxic damage and death of neuronal cells, epileptiform seizures, as well as the pathogenesis of different neurodegenerative disorders. To date, there is no information available on subtypes of the human glutamate metabotropic receptor (hmGluR), for example on its amino acid sequence or tissue distribution. This lack of knowledge particularly hinders the search for human therapeutic agents capable of specifically influencing any disorder attributable to a defect in the glutamatergic system. In view of the physiological potential and pathological significance of metabotropic glutamate receptors, there is a need for subtypes of human receptors and for cells that produce such subtypes in sufficient quantity to elucidate the electrophysiological and pharmacological properties of these proteins. For example, drug selection assays require actively purified human receptor proteins, which have not yet been achieved.

An objective of the present invention is to satisfy this need, that is to provide different subtypes of hmGluR, nucleic acids that encode them and host cells that produce such subtypes. In particular, the present invention discloses the subfamily of hmGluR which includes the subtype designated as hmGluR4, and the individual proteins of said subfamily. Hereinafter, said subfamily will be referred to as the hmGluR4 subfamily. Contrary to other subtypes of hmGluR, members of this subfamily are potently activated by L-2-amino-4-phosphobutyric acid (AP4) and, when expressed for example in Chinese hamster ovary (CHO) cells or in kidney cells of a hamster calf (BHK), negatively coupled to adenylate cyclase through protein G. Using a system comprising a subtype of recombinant hmGluR of the invention in the selection for drugs reactive to hmGluR offers ( among others) the possibilities of reaching a greater number of receptors per cell producing greater reagent performance and a higher signal-to-noise ratio in the tests as well as a greater specificity of the receptor subtype (potentially resulting in greater biological specificity and by disease).

The present invention further discloses a subtype of hmGluR characterized in that its amino acid sequence is approximately more than 65% identical to the sequence of the subtype of hmGluR4 having the amino acid sequence described in SEQ ID NO: 2.

According to the invention, the expression "subtype of hmGluR" it refers to a purified protein that belongs to the class of receptors coupled to the G protein and that by binding a glutamatergic ligand transduces an extracellular signal through a second intracellular messenger system. In such a case, a subtype of the invention is characterized in that it modifies the level of a cyclic nucleotide (cAMP, cGMP). Alternatively, signal transduction may occur through direct interaction of the G protein coupled to a receptor subtype of the invention with another membrane protein, such as an ionic channel or other receptor. A receptor subtype of the invention is believed to be encoded by a different gene that does not encode.

WO9210583 discloses the identification, isolation and purification of metabotropic glutamate receptors coupled to rat G protein and antibodies thereof. Nakajima et al (1993) JBC, 268, 11868-11773 discloses the cloning and molecular characterization of a metabotropic glutamate receptor from a rat retinal cDNA library, another subtype of metabotropic glutamate receptor. A particular subtype of the invention can be characterized by its different physiological profile, preferably by its signal transduction and pharmacological properties. Pharmacological properties are, for example, selectivity for agonist and antagonistic responses.

As defined herein, a glutamatergic ligand is for example L-glutamate or another compound that interacts with, and particularly that binds to, a subtype of hmGluR in a form such as glutamate, such as ACPD (1S acid, 3R- 1-aminocyclopentane-1,3-dicarboxylic acid), a ligand such as ACPD, for example, QUIS (quecuacuate), AP4, and the like. Other ligands, for example (R, S) -a-methylcarboxyphenylglycine (MCPG) or a-methyl-L-AP4, can interact with a receptor of the invention such that binding of the glutamatergic ligand is avoided.

As used here before or later, the terms "purified" or "isolated" they refer to a molecule of the invention in pure or enriched form that can be obtained from a natural source or by means of genetic engineering. The purified proteins, DNAs and RNAs of the invention can be useful in ways in which proteins, DNAs and RNAs as they occur in nature are not, such as the identification of compounds that selectively modulate the expression or activity of an hmGluR of the invention.

The purified hmGluR of the invention means a member of the hmGluR4 subfamily that has been identified and is free of one or more components of its natural environment. The purified hmGluR includes purified hmGluR of the invention in a recombinant cell culture. The enriched form of a subtype of the invention refers to a preparation containing said subtype in a higher than natural concentration, for example a fraction of a cell membrane containing said subtype. If said subtype is in pure form it is substantially free of other macromolecules, particularly of naturally occurring protein contaminants. If desired, the subtype of the invention can be solubilized. A preferred purified subtype of hmGluR of the invention is a recombinant protein. Preferably, the subtype of the invention is in an active state which means that it has both ligand binding and signal transduction activity. The activity of the receptor is measured according to methods known in the art, for example using a binding assay or a functional assay, for example an assay such as that described below.

The hmGluR subtypes of the hmGluR4 subfamily include the hmGluR4, hmGluR7 and hmGluR6 subtypes. Here a hmGluR4 subtype protein having the amino acid sequence set forth in SEQ ID NO: 2 is disclosed; a protein of the hmGluR7 type includes a polypeptide selected from the group consisting of the polypeptides having the amino acid sequences described in SEQ ID Nos: 4, 6, 8 and 10, respectively. The subtypes of hmGluR7 have the amino acid sequences set forth in SEQ ID Nos: 12 and 14, respectively. The hmGluR6 type protein according to the invention includes a polypeptide having the amino acid sequence described in SEQ ID NO: 16.

Additionally, variants of the receptor subtypes are disclosed here. For example, a variant of a subtype of hmGluR is a functional or immunological equivalent of that subtype. A functional equivalent is a protein, particularly a human protein, which shows a physiological profile essentially identical to the characteristic profile of said particular subtype. The in vitro and in vivo physiological profile includes an effector function of the receptor, electrophysiological and pharmacological properties, for example selective interaction with agonists or antagonists. Examples of functional equivalents may be splicing variants encoded by mRNA generated by the alternative splicing of a primary transcript, amino acid mutants and glycosylation variants. An immunological equivalent of a particular subtype of hmGluR is a protein or peptide capable of generating antibodies specific for said subtype. Portions of the extracellular domain of the receptor, for example peptides consisting of at least 6 to 8 amino acids, particularly 20 amino acids, are considered particularly useful immunological equivalents.

Additional variants included herein are soluble and membrane bound fragments and covalent or aggregation conjugates with other structural chemical units, these variants showing one or more receptor functions, such as ligand binding or signal transduction. Examples of hmGluR subtype fragments are polypeptides having the amino acid sequences set forth in SEQ ID Nos: 4, 6, 8, 10 and 16, respectively. The fragments of the invention have the amino acid sequence of SEQ ID No: 16 and can be obtained from a natural source, by chemical synthesis or by recombinant techniques. Due to their ability to compete with the endogenous counterpart of a subtype of hmGluR as described herein or its endogenous ligand, fragments, or derivatives thereof, which include the ligand binding domain are conceived as therapeutic agents.

Covalent derivatives include for example esters or aliphatic amides of a carboxylic receptor group, O-acyl derivatives of the hydroxyl group containing residues and N-acyl derivatives of the residues containing

to the amino group. Such derivatives can be prepared by linking functional groups with reactive groups found in the side chains and at the N and C terminals of the receptor protein. The protein of the invention can also be labeled with a group that can be detected, for example radiolabelled, covalently linked to rare earth chelates or conjugated to a fluorescent structural unit.

Additional derivatives are covalent conjugates of a protein of the invention with another protein or peptide (fusion proteins). Examples are fusion proteins that contain different portions of different glutamate receptors. Such fusion proteins may be useful for changing the coupling with G proteins and / or improving the sensitivity of a functional assay. For example, in such fusion proteins or chimeric receptors, the intracellular domains of a subtype of the invention can be replaced with the corresponding domains of another subtype of mGluR, particularly another subtype of hmGluR, for example a subtype of hmGluR belonging to another subfamily. . Particularly suitable for the construction of such a chimeric receptor are the intracellular domains of a receptor that activates the phospholipase C / Ca2 + signaling pathway, for example mGluR1 (Masu et al., Nature 349, 760-765) or mGluR5. An intracellular domain suitable for such an exchange is for example the second intracellular loop, also referred to as i2 (Pin et al., EMBO J. 13, 342-348 (1994)). In this way it is possible to analyze the interaction of a test compound with a ligand binding domain of a receptor of the invention using a calcium ion assay. The chimeric receptor according to the invention can be synthesized by means of recombinant techniques or agents known in the art that are suitable for protein cross-linking.

Aggregative derivatives are for example adsorption complexes with cell membranes.

In another embodiment, the present invention relates to a composition of matter that contains a subtype of hmGluR6 of the invention that includes a polypeptide having the amino acid sequence set forth in SEQ ID No: 16.

The protein of the invention is useful, for example, as an immunogen, in drug selection assays, as a reagent for immunoassays and in purification methods, such as for affinity purification of a binding ligand.

The protein of the invention can be obtained from a natural source, for example by isolation of brain tissue, by chemical synthesis or by recombinant techniques.

The invention further provides a method for preparing a subtype of hmGluR6 of the invention characterized in that suitable host cells that produce a receptor subtype of the invention are multiplied in vitro or in vivo. Preferably, the host cells are transformed (transfected) with a hybrid vector containing an expression cassette that includes a promoter and a DNA sequence encoding said cuto subtype DNA is controlled by said promoter. Subsequently, the hmGluR subtype of the invention can be recovered. Recovery comprises, for example, isolation of the subtype of the invention from host cells or isolation of host cells containing the subtype, for example from the culture broth. Particularly preferred is a method for the preparation of a functionally active receptor.

HmGluR muteins can be produced from a DNA encoding a protein of the hmGluR6 type of the invention whose DNA has been subjected to in vitro mutagenesis resulting in for example an addition, exchange and / or deletion of one or more amino acids. For example, substitution, deletion and insertion variants of a subtype of hmGluR of the invention are prepared by recombinant methods and selected by cross-immunoreactivity with the native forms of hmGluR.

A protein of the invention can also be subjected to in vitro shunt according to conventional methods known in the art.

Suitable host cells include eukaryotic cells, for example animal cells, plant and fungal cells, and prokaryotic cells, such as gram-positive and gram-negative bacteria, for example E. coli. Preferred eukaryotic host cells are of amphibious or mammalian origin.

As used herein, in vitro means ex vivo, including in this way, for example, cell culture and tissue culture conditions.

This invention further covers an isolated nucleic acid (DNA, RNA) that includes a purified, preferably recombinant nucleic acid (DNA, RNA) encoding a type of hmGluR6 of the invention, or a fragment of such a nucleic acid. In addition to being useful for the production of the recombinant hmGluR proteins above, these nucleic acids are useful as probes, thus easily allowing those skilled in the art

Identify and / or isolate nucleic acid encoding a protein hmGluR protein of the invention. The nucleic acid may be labeled or unlabeled with a detectable structural unit. In addition, the nucleic acid according to the invention is useful, for example, in a method for determining the presence of hmGluR, said method comprising the hybridization of the DNA (or RNA) encoding (or complementary to) hmGluR to analyze a nucleic acid sample and to determine the presence of hmGluR.

The purified nucleic acid encoding the hmGluR6 type protein of the invention includes nucleic acid that is free of at least one contaminating nucleic acid with which it is ordinarily associated in the natural source of nucleic acid for hmGluR. The purified nucleic acids are therefore present in a different form.

or arrangement in which it is found in nature. However, the purified nucleic acid for hmGluR encompasses the nucleic acid for hmGluR in cells that ordinarily express hmGluR where the nucleic acid is at a location on the chromosome different from that of the natural cells or is flanked by a different DNA sequence that that found in nature.

In particular, the invention provides an isolated or purified DNA molecule encoding a subtype of hmGluR6 of the invention, or a fragment of such DNA. By definition, such DNA includes a single coding DNA, a double stranded DNA consisting of said coding DNA and complementary DNA thereof, or this complementary (single stranded) DNA itself. Preferred is a DNA encoding the preferred subtypes of hmGluR mentioned above, or a fragment thereof. In addition, the invention relates to a DNA that includes such DNA.

Also disclosed herein is a DNA encoding a subtype of hmGluR4 or a portion thereof, particularly a DNA encoding the subtype of hmGluR4 having the amino acid sequence set forth in SEQ ID NO: 2, for example the DNA with the sequence of nucleotides set forth in SEQ ID NO: 1. An example of a DNA fragment encoding a portion of hmGluR4 is the portion encoding hmGluR4 of the cmR20 cDNA as described in the Examples.

Also disclosed here is a DNA encoding the subtype of hmGluR7, particularly a DNA encoding any of the subtypes of hmGluR7 that has the amino acid sequence set forth in SEQ ID Nos: 12 and 14, respectively, for example the DNA with the sequence of nucleotides set forth in SEQ ID Nos: 11 and 13, respectively. Additionally, a DNA fragment encoding a portion of a subtype of hmGluR7 is disclosed, particularly subtypes of hmGluR7 identified as preferred above. Examples of DNA fragments for hmGluR7 include the cmR2, cmR3, cmR5 and cR7PCR1 portions of cDNA encoding hmGluR7, as described in the Examples, or a DNA fragment encoding substantially the same amino acid sequence as that encoded by the portion of plasmid cmR2 encoding hmGluR7 deposited in the DSM on September 13, 1993, under the accession number DSM 8550. These DNAs encode portions of putative splicing variants of the hmGluR7 subtype described herein.

In particular, the invention provides a DNA encoding a subtype of hmGluR6 or a portion thereof, particularly a DNA encoding the subtype portion of hmGluR6, the amino acid sequence described in SEQ ID NO: 16, or a DNA encoding substantially the same amino acid sequence as that encoded by the portion of plasmid cmR1 encoding the hmGluR6 deposited in the DSM on September 13, 1993, under the accession number DSM 8549. An example of a DNA sequence is set forth in SEQ ID NO:

fifteen.

The nucleic acid sequences provided herein can be used to identify DNAs that encode additional subtypes of hmGluR. For example, nucleic acid sequences of the invention can be used to identify DNAs encoding additional subtypes of hmGluR belonging to the subfamily that includes hmGluR4. A method of identifying such DNA comprises contacting human DNA with a nucleic acid probe described above and identifying the DNA (s) that hybridize to that probe.

Examples of nucleic acids of the invention may alternatively be characterized as those nucleic acids encoding a subtype of the hmGluR of the invention and hybridizing under highly stringent conditions 1 to a DNA sequence set forth in SEQ ID Nos. 1 or 15, or a portion selected (fragment) from said DNA sequence. For example, selected fragments useful for hybridization are the portions of said DNA encoding proteins.

Hybridization stringency refers to conditions under which polynucleic acid hybrids are stable. Such conditions are evident to those ordinarily trained in this field. As those skilled in the art know, the stability of the hybrids is reflected in the melting temperature (Tm) of the hybrid that decreases approximately 1 to 1.5˚C with each 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is carried out under conditions of greater stringency, followed by washings of different stringency.

As used herein, high stringency refers to conditions that allow hybridization only of those nucleic acid sequences that form stable hybrids in Na + 1 M at 65-68 ° C. Highly stringent conditions can be supplied, for example, by hybridization in an aqueous solution containing 6x SSC, 5x Denhardt solution, 1% SDS (sodium dodecyl sulfate), Na + 0.1 pyrophosphate and 0.1 mg / ml of denatured salmon sperm DNA as a non-specific competitor. After hybridization, high stringency washings can be done in several stages, with a final wash (approximately 30 min) at the hybridization temperature in 0.2-0.1 x SSC, 0.1% SDS.

Moderate stringency refers to conditions equivalent to hybridization in the solution described above but at approximately 60-62 ° C. In that case the final wash is carried out at the hybridization temperature in 1x SSC, 0.1% SDS.

Low stringency refers to conditions equivalent to hybridization in the solution described above at 50 52 ° C. In that case, washing is carried out at the hybridization temperature in 2x SSC, 0.1% SDS.

It is understood that these conditions can be adapted and duplicated using a variety of dampers, for example formamide-based dampers, and temperatures. Denhardt and SSC's solutions are well known to those skilled in the art as well as other suitable hybridization buffers (see, for example, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA, or Ausubel, FM, et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, USA). The optimal hybridization conditions have to be determined empirically, since the length and GC content of the probe also play a role.

Given the orientation of the present invention, the nucleic acids of the invention can be obtained according to methods well known in the art. The present invention also relates to a process for the preparation of such nucleic acids.

For example, a DNA of the invention can be obtained by means of chemical synthesis, by means of recombinant DNA technology or by means of polymerase chain reaction (PCR). Preparation by means of recombinant DNA technology may involve the selection of a suitable cDNA or a genomic library. A suitable method for preparing a DNA or of the invention comprises the synthesis of an amount of oligonucleotides, their amplification by means of PCR methods, and their splicing to produce the desired DNA sequence. Suitable libraries are commercially available, for example the libraries employed in the Examples, or they can be prepared from samples of neural or neuronal tissue, for example from hippocampal and cerebellum tissue, cell lines and the like.

For individual subtypes of the hmGluR (and splice variants) of the invention, the expression pattern in neural or neuronal tissue may vary. Therefore, in order to isolate cDNA encoding a particular subtype (or splice variant), it is convenient to select libraries prepared from different suitable tissues

or cells As a selection probe, a DNA or an RNA that contains substantially the entire coding region of a subtype of the hmGluR of the invention, or a suitable oligonucleotide probe based on said DNA can be employed. A suitable oligonucleotide probe (for selection involving hybridization) is a single stranded DNA or RNA that has a nucleotide sequence that includes at least 14 contiguous bases that are equal (or complementary to) to any of the 14 or more contiguous bases set forth in any of SEQ ID Nos: 1, or 15. The probe can be labeled with a chemical structural unit suitable for easy detection. The nucleic acid sequences selected as probes must be of sufficient length and sufficiently unambiguous so that false positive results are minimized.

Preferred regions from which probes are constructed include the 5 'and / or 3' coding sequences, predicted sequences for encoding ligand binding sites, and the like. For example, either the full-length cDNA clones disclosed herein or fragments thereof can be used as probes. Preferably, the nucleic acid probes of the invention are labeled with suitable labeling media for easy detection after hybridization. For example, a suitable labeling medium is a radioactive label. The preferred method of labeling a DNA fragment is by incorporating a-dATP labeled with 32P with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art. The oligonucleotides are usually labeled at the end with y-ATP labeled with 32P and polynucleotide kinase. However, other methods (eg non-radioactive) can also be used to label the fragment or oligonucleotide, including for example enzyme labeling and biotinylation.

After selecting the library, for example with a portion of DNA that substantially includes the entire sequence encoding hmGluR or a suitable oligonucleotide based on a portion of said DNA, positive clones are identified by detecting a hybridization signal ; the clones identified are characterized by restriction enzyme mapping and / or DNA sequence analysis, and then examined, for example by comparison with the sequences set forth herein, to determine if they include DNA encoding a

hmGluR complete (that is, if they include codons for initiation and termination of translation). If the selected clones are incomplete, they can be used to re-select the same or a different library to obtain overlay clones. If the library is genomic, then overlay clones can include exons and introns. If the library is a cDNA library, then the overlay clones will include an open reading frame. In both cases, complete clones can be identified by comparison with the DNA and deduced amino acid sequences provided herein.

In addition, for the purpose of detecting any abnormality of an endogenous subtype of hmGluR6 of the invention a genetic selection can be carried out using the nucleotide sequence of the invention as hybridization probes. Also, based on the nucleic acid sequences provided herein, antisense type therapeutic agents can be designed.

It is envisioned that the nucleic acid of the invention can be easily modified by means of nucleotide replacement, nucleotide suppression, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof. Such modified sequences can be used to produce a mutant subtype of hmGluR6 that differs from receptor subtypes found in nature. Mutagenesis can be predetermined (site specific) or random. A mutation that is not a silent mutation should not put sequences outside the reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures such as loops or hairpins.

The genomic cDNA or DNA encoding native or mutant hmGluR6 of the invention can be incorporated into vectors for further manipulation. In addition, the invention relates to a recombinant DNA that is a hybrid vector comprising at least one of the aforementioned DNA.

The hybrid vectors of the invention include an origin of replication or an autonomously replicating sequence, one or more dominant marker sequences and, optionally, expression control sequence, signal sequences and additional restriction sites.

Preferably, the hybrid vector of the invention includes a nucleic acid insert described above operatively linked to an expression control sequence, in particular those described hereinafter.

Vectors typically perform two functions in collaboration with compatible host cells. One function is to facilitate the cloning of the nucleic acid encoding the subtype of the hmGluR6 of the invention, that is to produce usable amounts of the nucleic acid (cloning vectors). The other function is to allow replication and expression of gene constructs in a suitable host, either by maintaining it as an extrachromosomal element or by integrating it into the host chromosome (expression vectors). A cloning vector includes DNA as described above, an origin of replication or an autonomous replication sequence, selectable marker sequences, and optionally, signal sequences and additional restriction sites. An expression vector additionally includes expression control sequences essential for transcription and translation of the DNA of the invention. Thus, an expression vector refers to a recombinant DNA construct, such as a plasmid, a phage, a recombinant virus or another vector that, after introduction into a suitable host cell, results in the expression of the DNA. cloned Suitable expression vectors are well known in the art and include those that are replicable in eukaryotic and / or prokaryotic cells.

Most expression vectors are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned into E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replication independently of the host cell chromosome. The DNA can also be amplified by insertion into the host genome. However, the recovery of the genomic DNA encoding hmGluR is more complex than that of the exogenously replicated vector because digestion with a restriction enzyme is required to cut the DNA for hmGluR. The DNA can be amplified by means of PCR and directly transferred into the host cells without any replication component.

Conveniently, the expression and cloning vector contain a selection gene also referred to as a selectable marker. This gene encodes a protein necessary for the survival or development of transformed host cells cultured in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typically, the selection genes encode proteins that confer resistance to antibiotics and other toxins, for example ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or provide critical nutrients that are not available in the middle of the complex.

Since the amplification of the vectors is conveniently done in E. coli, a conveniently included

E. coli genetic marker and an origin of E. coli replication. These can be obtained from E. coli plasmids, such as pBR322, the Bluescript vector or a pUC plasmid.

Selectable markers suitable for mammalian cells are those that allow the identification of competent cells to take the nucleic acid for hmGluR, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes that confer resistance to G418 or hygromycin. Transfectants of mammalian cells are placed under selection pressure such that only those transfectants that have absorbed and express the marker are adapted to survive.

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to nucleic acid for hmGluR. Such promoter can be inducible or constitutive. The promoters are operably linked to DNA encoding hmGluR by removing the promoter from the DNA source by digestion with the restriction enzyme and inserting the isolated sequence from the promoter into the vector. Both the native hmGluR promoter sequence and many heterologous promoters can be used to direct amplification and / or DNA expression for hmGluR. However, heterologous promoters are preferred, because they generally allow for greater transcription and higher yield of the expressed hmGluR compared to the native hmGluR promoter.

Promoters suitable for use with prokaryotic hosts include, for example, the ctalactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thus allowing the trained operator to operatively link them to the DNA encoding hmGluR, using linkers or adapters to supply any of the required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgamo sequence operably linked to the DNA encoding hmGluR.

The transcription of the HmGluR gene of vectors into mammalian host cells can be controlled by promoters compatible with host cell systems, for example promoters derived from virus genomes. Plasmids suitable for expression of a subtype of the hmGluR6 of the invention in eukaryotic host cells, particularly mammalian cells, are for example vectors containing the cytomegalovirus promoter (CMV), vectors containing the RSV promoter and vectors containing SV40 and vectors containing the MMTV LTR promoter. Depending on the nature of their regulation, promoters may be constitutive or may be regulated by experimental conditions.

The transcription of a DNA encoding a subtype of hmGluR6 according to the invention by higher eukaryotes can be increased by inserting an enhancer sequence into the vector.

The different DNA segments of the vector DNA are operatively linked, that is to say they are contiguous and are placed in a functional relationship with each other.

The construction of vectors according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, rearranged to size, and ligated again in the desired manner to generate the required plasmids. If desired, an analysis is carried out to confirm the correct sequences in the plasmids constructed in a manner known in the art. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and conducting analyzes to evaluate the expression and function of hmGluR are known to those skilled in the art. The presence, amplification and / or expression of genes can be measured directly in a sample, for example, by means of conventional Southern type transfers, Northern type transfers to quantify mRNA transcription, point transfers (DNA or RNA analysis ), in situ hybridization, using an appropriately labeled probe based on a sequence provided herein, binding assays, immunodetection and functional assays. Suitable methods include those described in detail in the Examples. Those skilled in the art will easily realize how these methods can be modified, if desired.

The invention further provides host cells capable of producing a subtype of the hmGluR6 of the invention and including heterologous (outer) DNA encoding said subtype.

The nucleic acids of the invention can be expressed in a wide variety of host cells, for example those mentioned above, which are transformed or transfected with an appropriate expression vector. The receptor of the invention (or a portion thereof) can also be expressed as a fusion protein. Recombinant cells can then be cultured under conditions whereby the protein (s) encoded by the DNA of the invention is (are) expressed.

Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, such as E. coli, for example E. coli K-12 strains, DH5a and HB 101, or Bacilli. Additional suitable host cells for vectors encoding hmGluR include eukaryotic microbes such as filamentous fungi or yeast, for example Saccharomyces cerevisiae. Upper eukaryotic cells include insect, amphibian and vertebrate cells, particularly mammalian cells, for example neuroblastoma cell lines or fibroblast derived cell lines. Examples of preferred mammalian cell lines are for example HEK 293 cells, CHO cells, CV1 cells, BHK cells, L cells, LLCPK-1 cells, GH3 cells, L cells and COS cells. In recent years the propagation of cells and vertebrates in culture (tissue culture) has become a routine procedure. The host cells mentioned in this application include cells in an in vitro culture as well as cells that are within a host animal.

Suitable host cells for expression of an active recombinant hmGluR6 type protein of the invention conveniently express endogenous or recombinant G proteins. Cells that produce, if anything, little endogenous metabotropic glutamate receptor are preferred. The DNA can be stably incorporated into the cells or it can be expressed transiently according to conventional methods.

Stably transfected mammalian cells can be prepared by transfecting cells with an expression vector having a selectable marker, and developing transfected cells under selective conditions for cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to control the efficiency of transfection.

To produce such cells stably or transiently transfected, the cells must be transfected with a sufficient amount of nucleic acid encoding hmGluR to form a hmGluR6 type protein of the invention. The precise amounts of DNA encoding the hmGluR6 type protein of the invention can be determined and empirically optimized for a particular cell and assay.

A DNA of the invention can also be expressed in non-human transgenic animals, particularly warm-blooded transgenic animals. Methods for producing transgenic animals, including mice, rats, rabbits, sheep and pigs, are known in the art and are disclosed, for example by Hammer et al. (Nature 315, 680-683, 1985). An expression unit that includes a DNA of the invention encoding a protein of the hmGluR6 type together with appropriately positioned expression control sequences, is introduced into fertilized ovule pronuclei. The introduction can be achieved, for example by means of microinjection. The integration of the injected DNA is detected, for example by analysis of DNA transfers from suitable tissue samples. It is preferred that the introduced DNA be incorporated into the germ line of the animal so that it is transmitted to the progeny of the animal. Preferably, a transgenic animal is developed by means of directed manipulation of a mutation to alter an hmGluR sequence. Such an animal is useful for example to study the role of a metabotropic receptor in metabolism.

In addition, a genetically engineered animal can be developed so that it does not express one or more target genes through the introduction of a mutation in the hmGluR sequence, thus generating an animal that no longer expresses the functional gene for hmGluR. Such a genetically engineered animal so that it does not express an objective gene is useful for example to study the role of the metabotropic receptor in metabolism methods to produce modified mice known in the art.

Host cells are transfected or transformed with the aforementioned expression or cloning vectors of this invention and cultured in a conventional nutrient medium modified conveniently to induce promoters, select transformants, or amplify the genes encoding the desired sequences. Heterologous DNA can be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by means of the technique of precipitation together with calcium phosphate, by means of electroporation or mediated by lipofectin. . Qualified operators in this field know numerous methods of transfection. A successful transfection is generally recognized when any indication of the operation of this vector in the host cell is presented. Transformation is achieved using standard techniques appropriate to the particular host cells used.

The incorporation of cloned DNA into a suitable expression vector, the transfection of eukaryotic cells with a plasmid vector or a combination of plasmid vectors, each encoding one or more different genes or with linear DNA, and the selection of transfected cells are well known. in art (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).

Transfected or transformed cells are cultured using culture media and methods known in the art, preferably under conditions, by which hmGluR encoded by DNA is expressed. The composition of suitable media is known to those skilled in the art, so that they can be easily prepared. Suitable culture media are also commercially available.

Although the DNA supplied herein can be expressed in any suitable host cell, for example those mentioned above, preferred for expression of the DNA encoding functional hmGluR eukaryotic expression systems, particularly mammalian expression systems, including commercially available systems and other known systems by those trained in art.

The DNA for the human protein of the mGluR6 type of the invention is ligated into a vector, and introduced into suitable host cells to produce transformed cell lines that express a particular subtype of the hmGluR6 of the invention, or specific combinations of subtypes. The resulting cell line can then be produced in sufficient quantity for reproducible qualitative and quantitative analysis of the effects of an agonist, antagonist or allosteric receptor modulator. Additionally, mRNA can be produced by means of in vitro transcription of a DNA encoding a subtype of the invention. This mRNA can be injected into Xenopus oocytes where mRNA directs the synthesis of the active receptor subtype. Alternatively, the DNA encoding the subtype can be injected directly into oocytes. Transfected mammalian cells or injected oocytes can then be used in a drug selection test provided hereinafter. Such drugs are useful in diseases associated with pathogenesis of a subtype of hmGluR6 of the invention. Such diseases include diseases resulting from excessive glutamate action preferentially mediated by hmGluR, such as fulminant attack, epilepsy and chronic neurodegenerative diseases. Particularly useful for evaluating the specific interaction of compounds with specific subtypes of the hmGluR6 of the invention are stably transfected cell lines expressing an hmGluR of the invention.

Therefore, host cells expressing an hmGluR 6 protein of the invention are useful for drug selection and it is a further objective of the present invention to provide a method for the identification of a compound or signal that modulates the activity of hmGluR, said method comprising exposing the cells containing heterologous DNA encoding the hmGluR6 type protein of the invention, wherein said cells produce functional hmGluR, at least one compound or signal whose ability to modulate the activity of said hmGluR is intended to be determined, and after that, control in said cells the changes caused by said modulation. Such an assay allows the identification of agonist, antagonist and allosteric modulators of an hmGluR of the invention.

In a further aspect, the invention relates to an assay for the identification of compounds that modulate the activity of a subtype of the hmGluR6 of the invention, said assay comprising:

-

 contacting cells expressing an active subtype of the hmGluR of the invention and containing heterologous DNA encoding said subtype of hmGluR6 with at least one compound that is analyzed for its ability to modulate the activity of said receptor, and

-

analyze the cells for a difference in the level of the second messenger or receptor activity.

In particular, the invention relates to an assay for identifying compounds that modulate the activity of a subtype of the hmGluR of the invention, said assay comprising:

-

contacting cells expressing active protein of the hmGluR6 type of the invention and containing heterologous DNA encoding said subtype of hmGluR6 with at least one compound that is analyzed for its ability to modulate the activity of said receptor, and

-

 control said cells by a resulting change in the activity of the second messenger.

The result obtained in the test is compared with a suitable test as a negative control.

Test methods generally require comparison with different controls. A change in the activity of the recipient or in the level of the second messenger is said to be induced by a test compound if such an effect does not occur in the absence of the test compound. An effect of a test compound or a receptor subtype of the invention is said to be mediated by said receptor if this effect is not observed in cells that do not express the receptor.

As used herein, a compound or signal that modulates the activity of a protein of the hmGluR6 type of the invention refers to a compound or signal that alters the path of the response mediated by said hmGluR within a cell (compared to the absence of said hmGluR). A response pathway is activated by means of an extracellular stimulus, resulting in a change in the concentration of the second messenger or in the activity of the enzyme, or resulting in a change in the activity of a membrane bound protein, such as a receptor or ion channel. A variety of response pathways can be used, including, for example, the adenylate cyclase response pathway, the intracellular calcium phospholipase C / ion ion response pathway, or the coupling to an ion channel. Assays to determine the activity of adenylate cyclase are well known in the art, and

they include, for example, the trials disclosed by Nakajima et al., J. Biol. Chem. 267, 2437-2442 (1992)).

Thus, cells expressing the hmGluR6 type protein of the invention can be used for the identification of compounds, particularly low molecular weight molecules capable of acting as agonists.

or glutamate antagonists. Low molecular weight molecules of less than 1,000 Daltons are preferred. Within the context of the present invention, it is understood that an agonist refers to a molecule that is capable of interacting with a receptor, thus mimicking the action of L-glutamate. In particular, a glutamate agonist is characterized by its ability to interact with a protein of the hmGluR6 type of the invention, and therefore increase or decrease the stimulation of a response pathway within a cell. For example, an agonist increases or decreases a measurable parameter within the host cell, such as the concentration of a second messenger, just as the natural ligand ligand increases or decreases said parameter. For example, in a suitable test system, wherein the hmGluR6 type protein the invention is negatively coupled to adenylate cyclase, for example CHO or BHK cells expressing an hmGluR of the invention, such an agonist is capable of modulating the function of said hmGluR such that the intracellular concentration of cAMP is decreased.

In contrast, in situations where it is desirable to reduce the activity of hmGluR, antagonizing molecules are useful. Within the context of the present invention, it is understood that an antagonist refers to a molecule that is capable of interacting with a receptor or with L-glutamate, but that does not stimulate a response pathway within a cell. In particular, glutamate antagonists are generally identified by their ability to interact with a protein of the hmGluR6 type of the invention, and therefore reduce the ability of the natural ligand to stimulate a response pathway within a cell, for example interfering with L-glutamate binding with a hmGluR6 type protein of the invention or inhibiting other cellular functions required for hmGluR activity. For example, in a suitable assay, for example an assay involving CHO or BHK cells expressing a subtype of hmGluR6 type of the invention, a glutamate antagonist is capable of modulating the activity of a hmGluR6 type protein of the invention. such that the ability of the natural ligand to decrease the intracellular concentration of cAMP is weakened. Yet another alternative to achieve an antagonistic effect is to rely on the overexpression of antisense RNA for hmGluR. An agonist or antagonist that acts selectively on a hmGluR6 protein subfamily receptor, for example, hmGluR6, is preferred. Particularly useful is an agonist or antagonist that specifically modulates the activity of a particular subtype of hmGluR without affecting the activity of any other subtype.

An allosteric modulator of a protein of the hmGluR6 type of the invention interacts with the receptor protein in a different place from that of L-glutamate, thus acting as an agonist or antagonist. Therefore, the screening assays described herein are also useful for detecting an allosteric modulator of a receptor of the invention. For example, an allosteric modulator that acts as an agonist can improve the specific interaction between a hmGluR6 type protein of the invention and L-glutamate. If an allosteric modulator acts as an antagonist, it can for example interact with the receptor protein in such a way that the agonist binding is functionally less effective.

An in vitro assay for a glutamate agonist or antagonist may require that an hmGluR6 type protein of the invention be produced in sufficient quantities in a functional manner using recombinant DNA methods. An assay is then designed to measure a functional property of the hmGluR protein, for example the interaction with a glutamatergic ligand. The production of a hmGluR6 type protein of the invention is considered to occur in sufficient amounts, if the activity of said receptor results in a response that can be measured.

For example, mammalian cells, for example HEK293 cells, L cells, CHO-K1 cells, LLCPK-1 cells or GH3 cells (available for example from the American Tissue Type Culture Collection) are adapted to grow in a reduced glutamate medium, preferably glutamate free. An hmGluR expression plasmid, for example a plasmid described in the Examples, is transiently transfected into cells, for example by precipitation with calcium phosphate (Ausubel, FM, et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, USA). Cell lines stably expressing a protein of the hmGluR6 type of the invention can be regenerated for example by means of lipofectin-mediated transfection with hmGluR expression plasmids and a plasmid containing a selectable marker gene, for example pSV2-Neo (Southern and Berg, J. Mol. Appl. Genet. 1, 327-341 (1982)), a plasmid vector encoding the G-418 resistance gene. The cells that survive the selection are isolated and cultured in the selection medium. Resistant clonal cell lines are analyzed, for example by immunoreactivity with subtype-specific hmGluR antibodies or by assays for hmGluR functional responses after agonist addition. The cells that produce the desired subtype of hmGluR are used in a method to detect compounds that bind to a protein of the hmGluR6 type of the invention or in a method to identify a glutamate agonist or antagonist.

In a further embodiment, the invention provides a method for identifying compounds that bind to a subtype of hmGluR6, said method comprising the use of a subtype of hmGluR6 of the invention in a competitive binding assay. The underlying principle of a competitive binding essay is generally known in the art. In summary, the binding assays according to the invention are carried out allowing the compound to be analyzed for its hmGluR binding ability to compete with a known glutamatergic ligand, suitably labeled by the binding site in the target molecule. from hmGluR. A suitable labeled ligand is for example a radioactively labeled ligand, such as [3 H] glutamate, or a ligand that can be detected by its optical properties, such as absorbance or fluorescence. After removing the unbound ligand and analyzing in the compound the amount of unlabeled ligand bound to the hmGluR is measured. If the amount of labeled ligand is reduced in the presence of the test compound, it is said that this compound is bound to the target molecule. A competitive binding assay can be carried out, for example, with transformed or transfected host cells expressing an hmGluR of the invention or a membranous cell fraction containing a protein of the hmGluR6 type of the invention.

The compound bound to the target hmGluR can modulate the functional properties of the hmGluR and can therefore be identified as a glutamate agonist or antagonist in a functional assay.

Functional assays are used to detect a change in the functional activity of a protein of the hmGluR6 type of the invention, ie to detect a functional response, for example as a result of the interaction of the compound that is analyzed with said hmGluR. A functional response is for example a change (difference) in the concentration of a second relevant messenger, or a change in the activity of another membrane bound protein influenced by the receptor of the invention in cells expressing a functional hmGluR of the invention. (compared to a negative control). Those skilled in the art can easily identify a suitable assay to detect a change in the level of a second intracellular messenger indicative of the expression of an active hmGluR (functional assay). Examples include cAMP assays (see, for example, Nakajima et al., J. Biol. Chem. 267, 2437-2442 (1992), cGMP assays (see, for example, Steiner et al., J. Biol. Chem. 247, 1106 1113 (1972)), phosphatidyl inositol (PI) rotation assays (Nakajima et al., J. Biol. Chem. 267, 2437-2442 (1992)), calcium ion flow assays (Ito et al ., J. Neurochem. 56, 531-540 (1991)), arachidonic acid release assays (see, for example, Felder et al., J. Biol. Chem. 264, 20356-20362 (1989)), and the like. .

More specifically, according to the invention, a method of detecting a glutamate agonist comprises the steps of (a) exposing a compound to a subtype of the hmGluR6 of the invention coupled to a response route, under conditions and for a time sufficient to allow the interaction of the compound with the receptor and an associated response through the route, and (b) detect an increase or decrease in the stimulation of the response route resulting from the interaction of the compound with the subtype of hmGluR6, in relation to in the absence of the compound analyzed and from there determine the presence of a glutamate agonist.

A method of identifying a glutamate antagonist comprises the steps of (a) exposing a compound in the presence of a known glutamate agonist to a subtype of the hmGluR6 of the invention coupled to a response route, under conditions and for a time sufficient to allow the interaction of the agonist with the receptor and an associated response through the route, and (b) detect an inhibition of the stimulation of the response route by the agonist resulting from the interaction of the compound with the subtype of hmGluR6, in relation to Stimulation of the response route by the glutamate agonist alone and from there determine the presence of a glutamate antagonist. Inhibition can be detected, for example if the test compound competes with the glutamate agonist for the subtype of the hmGluR6 of the invention. The compounds that can be selected using such a method are for example blocking antibodies that specifically bind to the subtype of hmGluR6. In addition, such an assay is useful for the selection of compounds that interact with L-glutamate, for example soluble hmGluR fragments that contain part or all of the ligand binding domain.

Preferably, the interaction of an agonist or antagonist with a subtype of the hmGluR6 of the invention denotes binding of the agonist or antagonist with said hmGluR6.

As used herein, sufficient conditions and time for interaction of a glutamate agonist or antagonist candidate with the receptor will vary with the source of the receptor, however, the conditions generally suitable for binding occur between approximately 4˚C and approximately 40 ˚C, preferably approximately between 4˚C and approximately 37˚C, in a NaCl buffer solution between 0 and 2M, preferably between 0 and 0.9M, 0.1M NaCl being particularly preferred, and within a pH range between 5 and 9, preferably between 6.5 and 8. A sufficient time for binding and the response will generally be approximately between 1 ms and approximately 24 h after exposure.

In one embodiment of the present invention, the response route is a membrane linked adenylate cyclase route, and, for an agonist, the detection step comprises measuring a reduction or increase, preferably a reduction, in cAMP production. by the adenylate cyclase response path linked to

the membrane, in relation to the production of cAMP in the relevant control configuration. For the purpose of the present invention, it is preferred that the reduction or increase in cAMP production be equivalent to or greater than the reduction and / or increase induced by the L-glutamate applied at a concentration corresponding to its IC50 concentration. For an antagonist, the detection step comprises the measurement in the presence of the antagonist of a lower L-glutamate that induced a decrease or increase in cAMP production by the membrane bound adenylate cyclase response path, compared to the production of cAMP in the absence of the antagonist. The cAMP measurement can be carried out after the destruction of the cell or by means of a cAMP-sensitive molecular probe loaded into the cell, such as a fluorescent dye, which changes its properties, for example its fluorescence properties, by cAMP binding.

The production of cyclic AMP can be measured using methods well known in the art, including for example, the methods described by Nakajima et al., See above, or using commercially available kits, for example kits that include radioactively labeled cAMP, for example [125I] cAMP or [3H] cAMP. Examples of kits are the Amersham Scintillation Proximity Assay Kit, which measures CAMP production by iodinated cAMP competition with cAMP antibodies, or the Amersham Cyclic AMP [3H] Assay Kit.

In assay systems using cells that express receptor subtypes that are negatively coupled with the adenylate cyclase pathway, that is to say that it causes a decrease in cAMP after stimulation and an increase in cAMP after reduction of stimulation, it is preferred to expose the cells to a compound that reversibly or irreversibly stimulates adenylate cyclase, for example forskolin, or that is a phosphodiesterase inhibitor, such as isobutylmethylxanthine (IBMX), before the agonist or antagonist (potential) receptor is added.

In another embodiment of the invention, the response route is the Ca2 + mobilization route by PI hydrolysis. Such an assay to determine the specific interaction of a test compound with a subtype of the hmGluR6 of the invention may be functionally related to changes in the concentration of intracellular calcium (Ca2 +) ion. Different methods are known to determine a change in the intracellular concentration of Ca2 + in the art, for example a method that involves a fluorescent dye sensitive to calcium ion, such as fura-2 (see Grynkiewisz et al., J. Biol. Chem. 260, 3440-3450, 1985), fluo-3 or Indo-1, such as the QuinZ calcium fluorine method described by Charest et al. (J. Biol. Chem. 259, 8679-8773 (1993)), or the method of aequorin photoprotein described by Nakajima-Shimada (Proc. Natl Acad. Sci. USA 88, 6878-6882 (1991)). In one embodiment of the invention, the concentration of the intracellular calcium ion is measured by microfluorometry in recombinant cells loaded with fluorescent dyes sensitive to calcium fluo-3 or fura-2. These measurements can be carried out using cells grown on a coverslip allowing the use of an inverted microscope and video imaging technologies or a fluorescence photometer to measure calcium concentrations at the individual cellular level. For both approaches, cells transformed with a plasmid that expresses hmGluR have to be loaded with the calcium indicator. To this end, the culture medium is removed from the cells and replaced with a solution containing fura-2 or fluo-3. Cells are used for calcium measurements preferentially for the next 8 h. Microfluorometry follows standard procedures.

The Ca2 + signals that result from the functional interaction of compounds with the target molecule can be transient if the compound is applied for a limited period of time, for example through a perfusion system. Using a transient application, different measurements can be made with the same cells allowing internal controls and the analysis of a large number of compounds.

Functional coupling of a protein of the hmGluR6 type of the invention with Ca2 + signaling can be achieved, for example in CHO cells, by means of different methods:

(i)

 co-expression of a recombinant protein of the hmGluR6 type of the invention and a voltage-activated cationic channel, whose activity is functionally related to the activity of hmGluR;

(ii)

 expression of a chimeric hmGluR receptor, which directly stimulates the PI / Ca2 + pathway;

(iii) coexpression of a recombinant protein of the hmGluR6 type of the invention with a cationic channel that depends on recombinant Ca2 + permeable cAMP.

In other expression systems the functional coupling of an hmGluR with Ca2 + signaling can be achieved by transfection of an hmGluR of the invention if these cells naturally express (i) voltage activated Ca channels, whose activity is functionally related to the activity of mGluR or (ii) ion channels that depend on cAMP permeable to Ca2 +. For example, GH3 cells that naturally express voltage-activated Ca channels directly allow the application of Ca2 + assays to analyze the functional activity of hmGluR by cotransfection of hmGluR.

Cell-based screening assays can be designed, for example, through the construction of cell lines in which the expression of a reporter protein, that is an easy-to-assay protein, such as asa, chloramphenicol acetyltransferase (CAT) or Luciferase, depends on the function of a protein of the hmGluR6 type of the invention. For example, a DNA construct that contains a cAMP response element is operably linked to a DNA encoding luciferase. The resulting DNA construct comprising the DNA for the enzyme is stably transfected into a host cell. The host cell is then transfected with a second DNA construct that contains a first segment of DNA encoding a hmGluR6 type protein of the invention operably linked to additional segments of DNA necessary for receptor expression. For example, if the binding of a test compound with the hmGluR6 subtype of the invention results in high levels of cAMP, luciferase expression is induced or decreased, depending on the promoter chosen. Luciferase is exposed to luciferin, and photons emitted during the oxidation of luciferin by luciferase are measured.

The drug selection assays provided herein will allow the identification and design of specific compounds for the receptor subtype, particularly ligands that bind to the receptor protein, eventually leading to the development of a disease-specific drug. If they are designed for a very specific interaction only with a particular subtype of hmGluR6 (or a predetermined selection of the subtype of hmGluR6) such a drug is more likely to exhibit fewer adverse side effects than a drug identified by selection with cells that express a variety (unknown) of receptor subtypes. Also, assays of a single receptor subtype of the invention or specific combinations of different receptor subtypes with a variety of potential agonists or antagonists provide additional information regarding the function and activity of the individual subtypes and should lead to the identification and design of compounds that are capable of a very specific interaction with one or more receptor subtypes.

In another embodiment the invention provides polyclonal and monoclonal antibodies generated against a subtype of the hmGluR6 of the invention. Such antibodies may be useful for example for immunoassays including immunohistochemistry as well as diagnostic and therapeutic applications. For example, antibodies specific to the extracellular domain, or portions thereof, of a particular subtype of hmGluR6 can be applied to block the endogenous subtype of hmGluR6.

The antibodies of the invention can be prepared according to methods well known in the art using as an antigen a subtype of the hmGluR6 of the invention, a fragment thereof or a cell expressing said subtype or fragment. The antigen may represent the active or inactive form of the receptor of the invention. Antibodies may be able to distinguish between active or inactive form. Factors to consider in the selection of fragments of the subtype as antigens (either as a synthetic peptide or as a fusion protein) include antigenicity, accessibility (i.e. extracellular and cytoplasmic domains) and uniqueness with the particular subtype.

Particularly useful are antibodies that recognize and selectively bind to subtypes of the subfamily receptor described above without binding to a subtype of another subfamily and antibodies that selectively recognize and bind to a particular subtype without binding to any other subtype.

The antibodies of the invention can be administered to an individual who requires them using standard methods. Someone skilled in the art can easily determine dosage forms, treatment regimens etc., depending on the mode of administration employed.

The invention relates particularly to specific embodiments such as those described in the Examples that serve to illustrate the present invention but should not be considered as limiting thereto.

Abbreviations: hmGluR = human glutamate metabotropic receptor, nt = nucleotide

Example 1: cDNA encoding hmGluR4

The cDNA clones for human mGluR4 are isolated from human fetal brain and from human cerebellum cDNA libraries by means of low stringency hybridization using a radioactively labeled probe of rat mGluR4 generated by PCR from cDNA from brain of rat.

1.1 Preparation of rat (forebrain) poly (A) + RNA: Sprague-Dawley adult male rats are sacrificed by suffocation, their forebrains are removed and immediately frozen in liquid N2. Total RNA is isolated using the guanidinium thiocyanate procedure (Chomczynski and Sacchi (1987), Anal. Biochem. 162, 156 159). The enrichment of poly (A) + RNA is achieved by means of affinity chromatography on oligo (dT) -cellulose according to standard procedures (Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA).

1.2 Synthesis of the first cDNA chain by PCR: Poly (A) + RNA (mRNA) is reverse transcribed into DNA by Moloney Murid Leukemia Virus Reverse Transcriptase (M-MLV RT, BRL). 50 µl reactions are carried out as follows: 10 µg of poly (A) + rat forebrain RNA is heated in 10 µl of sterile H2O to 70 hasta C for 10 min and then quickly cooled on ice. Then, 10 µl 5x of reaction buffer (250 mM Tris-HCl pH 8.3, 375 mM KCI, 15 mM MgCl2), 5 µl 0.1 M dithiothreitol, 5 µl of mixed dNTP (dATP, dCTP, dGTP) are added , dTTP, 10 mM each of Pharmacia), 1.25 µl oligo-dT12-18 (2 mg / ml, Pharmacia), 2.5 µl RNA without (40 U / µl, Promega), 12.25 µl sterile H2O and 4 µl (200 U / µl) of M-MLV at RT. The reaction is carried out at 37˚C for 60 min.

1.3 PCR conditions for the generation of the rat mGluR4 fragment: The oligodeoxynucleotide initiators used for the PCR are synthesized by the phosphoramidite method. The sequences are listed in Table 1.

Table 1

P1: 5’- GTCAAGGCCTCGGGCCGGGA -3 ’

corresponding to pb 1921-1940 cDNA for rat mGluR4

[Tanabe et al., Neuron 8: 169-179 (1992)]

P2: 5’- CTAGATGGCATGGTTGGTGTA-3 ’

corresponding to pb 2788-2808 of the cDNA for rat mGluR4

[Tanabe et al., Neuron 8: 169-179 (1992)]

The conditions of a standard PCR of a 100 µl reaction mixture are: 30 ng rat forebrain cDNA, 50 pmol of each of the initiators P1 and P2, 200 µmol of each of the four deoxynucleoside triphosphates dATP, dCTP , dGTP and dTTP, 10% DMSO in PCR buffer (10 mM Tris-HCI, pH 8.3, 1.5 mM MgCl2, 50 mM KCI, 10 mM-mercaptoethanol, 0.05% Tween (w / v ), NP-40 0.05% (w / v)), and 0.5 U of AmpliTaq Polymerase (Perkin Elmer Cetus).

The amplification is carried out using the following conditions: 30 s of denaturation at 93˚C, 1 min 30 s of hybridization at 56˚C, and 3 min of extension at 72˚C, for a total of 40 cycles. Initial denaturation is carried out for 4 min at 94˚C.

1.4 Subcloning of the rat mGluR4 PCR fragment: Restriction endonuclease digestions, the use of modification enzymes, vector preparation (dephosphorylation, gel purification), ligaments, E. coli transformation, and plasmid DNA preparations are carried out according to standard procedures (Sambrook, et al. (1989), see above).

The PCR fragment (888 bp) obtained according to the procedure described in 1.3 is ligated into the Smal site of the Bluescript SK + plasmid (Stratagene, La Jolla, USA). The fragment inserted into the Bluescript vector is sequenced from both ends using the T7 and T3 primers (Stratagene, La Jolla, USA).

1.5 Preparation of a radioactively labeled probe: 20-50 ng of the rat mGluR4 fragment generated by PCR is gel purified and labeled with 32P by random priming using a DNA labeling kit (Boehringer Mannheim).

1.6 Selection of the cDNA library: approximately 1x106 phages are selected from a human fetal brain library (AZAPII Stratagene, La Jolla, USA), human hippocampus (AZAP, Stratagene, La Jolla, USA), and a cerebellum cDNA library human (AZAP, Stratagene) for hybridization with the rat mGluR4 fragment. Hybridization is carried out in 5x SSC, 0.02% Ficoll (w / v) (Type 400), 0.02% polyvinylpyrrolidone (w / v), 0.1% SDS (w / v), 50 µg / ml Herring Testis DNA. Prehybridization is carried out between 30 min and 3 hours at 58˚C. Hybridization is carried out at low stringency at 58 ° C overnight in the same solution that contains the 32P-labeled fragment at a concentration of 1-3 x 105 cpm / ml. They were washed three times for 20 min each time at 58˚C in 2x SSC / 0.1% SDS.

Phages that hybridize with the rat mGluR4 probe are purified by means of a second and third round of selection under the conditions described above. The cDNA inserts housed by purified phages are rescued by excision in vivo using the ExAssist / SOLR system (Stratagene, La Jolla,

E01124391 11-29-2011

USA)

1.7 Characterization of isolated cDNA clones: Different cDNA inserts are characterized by restriction enzyme mapping and DNA sequence analysis. One of these clones, cmR20 cDNA (isolated from a human cerebellum library) contains an insert of approximately 3.3 kb. The sequence analysis of cmR20 indicates that it contains almost the entire coding region of human mGluR4 that includes a translation termination codon (nt 158 to 2739, see SEQ ID NO: 1) as well as approximately 750 nt of region 3 'not translated. The 5 ’end is missing, which includes the translational start codon.

1.8 Isolation of the 5 ’end of human mGluR4: To complete the coding region of human mGluR4, PCR reactions are carried out using human genomic DNA or the first strand of human brain RNA cDNA as a template. The P3 sense primer corresponds to the 5 ′ end of the rat mGluR4 cDNA, the P4 antisense primer with nt 440-459 of the rat mGluR4 cDNA.

Table 2

P3: 5’-GCGCTGCAGGCGGCCGCAGGGCCTGCTAGGGCTAGGAGCGGGGG3 ’

corresponding to nt 11-37 of the rat mGluR4 cDNA

[Tanabe et al., Neuron 8: 169-179 (1992)]

P4: 5’-GCGGAATTCCCTCCGTGCCGTCCTTCTCG-3 ’

corresponding to nt 440-459 of the rat mGluR4 cDNA

[Tanabe et al., Neuron 8: 169-179 (1992)]

Additional sequences are underlined, sites for restriction enzymes are indicated in bold.

The PCR reactions for a 100 µl reaction mixture are: 400 ng of human genomic DNA, 1 µM of each initiator, 2 mM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP and dTTP) in PCR buffer (Tris-HCI 10 mM, pH 8.3, 1.5 mM MgCl2, 50 mM KCI, and 2 U of AmpliTaq Polymerase Amplification is carried out using the following conditions: 1 min of denaturation at 95˚C, 1 min of hybridization at 56 ˚C, and 1 min extension at 72˚C, for a total of 32 cycles.The initial denaturation is carried out for 3 min at 94˚C.

The products of different independent PCRs are digested with the restriction enzymes Pstl and EcoRI, gel purified, and ligated into the PstI / EcoRI sites of pBluescript SK (Stratagene). Subcloned fragments of different independent PCRs are analyzed by means of DNA sequence analysis (cR4PCR1-4). Sequence analysis reveals that clone cR4PCR2 encodes 380 nt of an hmGluR4 coding region that includes the translation initiation codon (nt 1- 380, see SEQ ID NO: 1). cR4PCR2 overlaps the 3 ’end for 223 nt with cmR20.

The complete amino acid deduced sequence of the hmGluR4 protein is set forth in SEQ ID NO: 2.

Example 2: cDNA clones encoding hmGluR7

The selection of human cerebellum and human fetal brain cDNA libraries by means of low stringency hybridization using a radioactively labeled mGluR4 fragment (as described in 1.5 and 1.6) allows the isolation of cDNA clones that identify to the subtype of human glutamate metabotropic receptor mGluR7. The characterization of cDNA clones by means of DNA sequence analysis reveals that the isolated cDNAs represent at least two apparent mRNA splicing variants for human mGluR7. The cmR2 cDNA clone (isolated from a human fetal brain cDNA library) is 3804 nt in size. The cmR2 clone containing 2604 nt of the hmGluR7 coding sequence includes a translation termination codon followed by 1200 nt of the 3 ′ untranslated sequence (see SEQ ID NO: 3). The cmR3 cDNA clone (isolated from a human hippocampus cDNA library) is 1399 nt in size (SEQ ID NO: 5). cmR3 containing 270 nt of the region encoding the 3 'end of hmGluR7 includes a translation termination codon (the deduced amino acid sequence is set forth in SEQ ID NO: 6) followed by 1129 nt of the 3' sequence no translated. The cmR3 sequence is completely contained in cmR2 but differs from cmR2 by the suppression of the 92 nucleotides that extend from nt at position 2534 to nt at position 2625 in SEQ ID NO: 3). This apparent splice variant of hmGluR7 generates at a 3 ′ end different from the amino acid sequence deduced from hmGluR7. The cmR5 cDNA clone (isolated from a human fetal brain cDNA library) is 1588 nt in size (SEQ ID NO: 7). The cmR5 cDNA clone overlaps 1424 nt with the cmR2 clone of

CDNA It diverges at the 3 ’end exactly at the insertion / suppression position of nt 92 of cmR2 / cmR3. An additional 164 nt of cmR5 encodes either intronic sequences as indicated by the presence of a splice donor sequence conserved immediately after the divergence site of the cmR5 and cmR2 / cmR3 sequence, or represent a third splice variant.

The region encoding the 5 'end of the DNA for hmGluR7 lost in the clones cmR2, cmR3, and cmR5 of cDNA, is isolated by a combination of a selection from a genomic library and PCR techniques. A Lambda-Fix genomic library (Stratagene) is screened with an EcoRI / Smal restriction fragment comprising nt 1 1304 of the cmR2 cDNA clone under conditions of high stringency hybridization as described in Sambrook, et al. (1989), see above. Lambda clones that hybridize with the 5 ′ end of the cmR2 cDNA clone are purified and analyzed by restriction analysis and DNA sequencing. The complete 5 ′ end of the coding region of human mGluR7 that includes the ATG translation initiation codon is amplified by PCR from human brain cDNA using primer sequences derived from cloned genomic fragments. The PCR fragments have a size of 557 nt. They are referred to as cR7PCR1 and are described as SEQ ID NO: 9. The deduced amino acid sequence is set forth in SEQ ID NO: 10. cR7PCR1 overlaps at the 3 'end with cmR2 for 392 nt.

The DNA sequences encoding the complete hmGluR7a and b proteins are set forth in SEQ ID Nos: 11 and 13, respectively. The deduced amino acid sequences are given in SEQ ID Nos: 12 and 14, respectively. Comparison of the deduced amino acid sequences reveals approximately 70% sequence identity with the hmGluR4 subtype of Example 1.

Example 3: cDNA encoding partial hmGluR6

An individual cDNA clone, cmR1, is isolated with a 1.0 kb insert from a human hippocampus library by means of low stringency hybridization using the hmGluR fragment described above in example 1.5 and 1.6. Approximately 630 nucleotides are homologous to human mGluR4. Additional sequences at the 5 'and 3' end of cmR1 apparently encode intronic sequences as indicated by the presence of putative sequences at the splice acceptor site and at the splice donor site. The cmR1 cDNA clone identifies a portion of the hmGluR6 human glutamate metabotropic receptor subtype (SEQ ID NO: 15). The deduced amino acid sequence is set forth in SEQ ID NO: 16.

The complete coding region of hmGluR6 is isolated by screening the cDNA and genomic libraries under conditions of high stringency with the cmR1 clone of cDNA as a probe. Comparison of the deduced amino acid sequences reveals approximately 70% sequence identity with the hmGluR4 of Example 1.

Example 4: Expression of hmGluR cDNAs in mammalian cells

4.1 Receptor expression plasmids: cDNAs encoding the previous proteins of hmGluR4, hmGluR6, and full-length hmGluR7 are generated from cDNA fragments and ligated into mammalian expression vectors based on constitutive promoters (CMV , SV40, RSV) or in inducible promoters. Examples are pBK-CMV (Stratagene), pBK-RSV (Stratagene), pCMV-T7 (Sibia, Inc.) and pICP4 (Novagen, USA).

The full-length cDNA encoding the subtype of hmGluR4 is incorporated into the mammalian expression vector pBK-CMV by linking the 5 'end fragment of hmGluR4 (clone cR4PCR2) with clone cmR20 of the cDNA at the unique Xhol site that is located in nt 346-351 of the cDNA for hmGluR4. Specifically, plasmid pBK-CMV-hmGluR4 is generalized by means of a three-way ligation of the NotI / XhoI fragment of cR4PCR2, of the XhoI / NotI fragment of the cmR20 cDNA clone and of the NotB-digested pBK-CMV vector. Plasmid pCMV-T7hmGluR4 is generated by means of a three-way ligation of the Pstl / Xhol fragment of cR4PCR4, the XhoI / EcoRI fragment of cmR20 and the vector pCMV-T7-2 digested with PstI / EcoRI. Both expression constructs contain the complete coding region of hmGluR4 as well as approximately 750 nt of the 3 ’untranslated sequences.

The full-length cDNAs that represent the two splice variant of hmGluR7, called hmGluR7a (SEQ ID NO: 12) and hmGluR7b (SEQ ID NO: 14), are incorporated into pCMV-T7-2 (SIBIA Inc.) using the clones which overlap cmR2, cmR3 and hcR7PCR1 of the cDNA. A full-length hmGluR7b expression construct, called pCMV-T7-hmGluR7b, is prepared by means of a three-way ligation of the PstI / BsaI fragment of hcR7PCR1, the BsaI / EagI fragment of cmR2 and the PstI / NotI of pCMVT7-2. Plasmid pCMV-T7-hmGluR7b contains the entire coding region of hmGluR7b and 191 nt of untranslated 3 'sequences. To construct a full-length hmGluR7a expression construct, called pCMVT7-hmGluR7a, a 370 bp HindIII / EagI fragment of cmR2 is exchanged with the corresponding cmR3 fragment. The BsaI / EagI fragment of the resulting clone is used for a three-way ligation as described above.

Plasmid pBK-CMV-hmGluR6 is generated analogously using conventional techniques (Sambrook et al., See above).

4.2 Transfection of mammalian cells: Mammalian cells (eg CHO-K1, GH3; American Tissue Type Culture Collection) are adapted to grow in glutamate free medium (modified Dulbecco Eagle medium lacking L-glutamate and containing a reduced concentration of 2 mM L-glutamine, supplemented with 0.046 mg / ml proline and 10% dialyzed fetal bovine serum, Gibco-BRL). HmGluR expression plasmids are transiently transfected into the cells by precipitation with calcium phosphate (Ausubel, F. M., et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, USA).

Cell lines that stably express hmGluR are generated by means of lipofectin-mediated transfection (Gibco-BRL) of CHOK1 cells with hmGluR and pSV2-Neo expression plasmids (Southern and Berg, 1982), a plasmid vector that encodes to the resistance gene G-418. The cells are cultured for 48 hours before the addition of 1 mg / ml of G-418 sulfate (Geneticina, Gibco). The medium is replaced every two or three days. Cells that survive selection with G-418 are isolated and cultured in the selection medium. 32 lines of clonal cells resistant to G-418 are analyzed six to eight weeks after initial transfection by hmGluR protein expression by means of immunoreactivity with the anti-hmGluR7 antibody (immunodetection, see 4.3, below) and responses functional after agonist addition through a radioimmunoassay with cAMP (see 5.1, below).

Similarly, the expression constructs of hmGluR pBK-CMV-hmGluR4, pCMV-T7-hmGluR4, pCMV-T7hmGluR7b and pCMV-T7-hmGluR7a are expressed transiently and stably in mammalian cells (CV1, CHO, HEK293 according to standard procedures (Ausubel, FM, et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, USA). Transfected cells are analyzed by the expression of hmGluR through different assays: [3 H] -glutamate binding studies, immunocytochemistry using antibodies specific for the subtype of hmGluR, and assays that detect a change in the intracellular concentration of cAMP ([ cAMP]).

4.3 Immunodetection of hmGluR protein expression with subtype-specific hmGluR antibodies: HmGluR protein expression is analyzed by immunocytochemistry with subtype-specific hmGluR antibodies (see Example 7). 1 to 3 days after transfection of the cells, they are washed twice with phosphate buffered saline (PBS), fixed with PBS / 4% para-formaldehyde for 10 min and washed with PBS. The cells are permeabilized with 0.4% PBS / Triton X-100, followed by a wash with 10 mM PBS / glycine, and PBS. The cells are blocked with PBSTB (1x PBS / 0.1% Triton X-100/1% BSA) for 1 h and subsequently incubated with immunopurified hmGluR antiserum (0.5-2.0 µg / ml in PBSTB) for 1 h. After three washes with PBS, the cells are incubated for 1 h with goat anti-rabbit IgG conjugated with alkaline peroxidase (1: 200 in PBSTB; Jackson Immuno Research). The cells are washed three times with PBS and immunoreactivity is detected with 0.4 mg / ml naphthosphosphate (Biorad) / 1 mg / ml Fast Red (Biorad) / 10 mM Levamisole (Sigma) / 100 mM Tris / HCl pH 8.8 / 10 mM NaCl / 50 mM MgCl2. The coloring reaction is stopped after 15 min, subsequently washing with PBS. 2 to 4 cell lines are identified, each expressing homogeneously hmGluR4, hmGluR6 or hmGluR7, by means of immunocolorization.

Example 5: Use of stable cell lines expressing hmGluR for the screening of receptor activity modulators

Stable cell lines expressing hmGluR4, hmGluR6 and hmGluR7 are used to screen allosteric agonists, antagonists and modulators. Such compounds are identified by binding studies that employ [3H] glutamate and / or the measurement of changes in intracellular levels of the second messenger ([cAMP], [Ca2 +]).

5.1 cAMP radioimmunoassay: Ligand binding and agonist-induced depression of forskolin-stimulated cAMP accumulation (changes in intracellular cAMP concentration) are analyzed by means of cAMP radioimmunoassay (Amersham). The cells are seeded in 12-well plates with a density of 0.5-2.0 x 105 cells per well and grow for 2 to 4 days until a confluent layer of cells is obtained. The cells are washed twice with PBS and incubated for 20 min in PBS containing 1 mM 3-isobutyl-1-methylxanthine (IBMX). The cells are incubated with fresh PBS containing 10 µM forskolin, 1 mM IBMX and a known hmGluR agonist for 20 min. The agonistic effect is stopped and the cAMP produced by the cells is released by adding 1 ml of a mixture of ethanol-water-HCl (100 ml of ethanol, 50 ml of water, 1 ml of 1 M HCI) then having aspirated the medium that contains the drug. CAMP levels are determined by means of a cAMP radioimmunoassay that involves [3H] cAMP (Amersham).

Subtypes 4, 6 and 7 of HmGluR are negatively coupled to adenylate cyclase when expressed in CHO cells. The binding of an agonist leads to an inhibition of forskolin-induced cAMP accumulation. All subtypes are sensitive to AP-4, which means that AP 4 has an agonistic effect at a concentration.

E01124391 11-29-2011

less than 1 mM.

5.2 Measurement of intracellular [Ca2 +]: Cells transformed with one of the above expression plasmids are loaded with a calcium sensitive fluorescent dye such as fura-2 or fluro-3. To achieve this, cells are plated in individual wells, individual wells containing a coverslip, or 96 well plates and grown for 1 to 5 days until a 50-100% confluent cell layer is obtained. The wells are washed three times with a balanced saline solution (BBS) and incubated for 1 h in BBS followed by three additional washes with BBS. The cells are then incubated for 20 to 60 min in a solution containing 50 µg of fura-2-AM (or fluro3-AM) (Molecular Probes, Inc.), 4.99 ml of BBS, 75 ml of DMSO and 6 , 25 pg of Pluronic (Molecular Probes, Inc). The cells are washed 3 times with BBS containing 2 mg / ml bovine albumin followed by three washes in BBS. After allowing the recovery of the cells for at least 10 min they are used for microfluorometric measurements of [Ca2 +].

The cells are transferred to an apparatus for fluorometry such as an inverted microscope, a fluorescence reader spectrofluorometer. The fluorescence of the calcium indicator (for example fura-2 or fluo-3) is induced by light illumination of a wavelength covered by the excitation spectrum of the dye (fura-2: 340/380 nm, fluoro- 3 3 480 nm). An increase in the intracellular concentration of the free calcium ion is observed as an increase in fluorescence of fura-2 or fluo-3 excited at 340 nm and 480 nm, respectively, or a decrease in fluorescence of fura-2 excited at 380 nm.

As a positive control, L-glutamate is applied at a concentration corresponding to its EC50 value on the cells, thereby inducing a measurable increase in the concentration of intracellular calcium ion. A test compound is said to be an agonist if it induces a Ca2 + signal comparable to that induced by glutamate. A test compound is said to be an antagonist if the calcium signal induced by glutamate is lower in the presence of the test compound than in the absence of the test compound.

Example 6: chimeric hmGluR4, receptors 6 and 7

The intracellular domains of mGluR1, particularly the second intracellular loop (i2) and the C-terminal region, have proven critical for the binding of G proteins, which activate the phospholipase C / Ca2 + signaling pathway, without changing the pharmacological profile of the receptor (Pin et al., EMBO J. 13, 342-348, (1994)). Conventional PCR mutagenesis techniques are used to exchange the intracellular domains of hmGluR 4,6, and 7 with the corresponding domains of hmGluR1. CHO stable cell lines are generated with hmGluR4 / 1, 6/1 and 7/1 chimeric expression constructs that allow the analysis of the influence of modulators of receptor activity (hmGluR 4, 6, 7) using assays that depend on Ca2 +. Next, the generation of a chimeric hmGluR7 / 1 receptor is described. Expression constructs with chimeric hmGluR4 / 1 and hmGluR6 / 1 are generated using analogous cloning and PCR techniques. (i) The pCMVhmGluR7b expression construct is digested with Eagl, thus releasing the entire cDNA insert. The cDNA is cloned into the Notl site of pBluescript-Not, a derivative of pBluescript II (Stratagene) where polylinker sequences between the unique sites Kpnl and Notl are suppressed. The resulting clone is referred to as pBluescript-Not-hmGluR7. (ii) The transmembrane region of hmGluR1 is cloned by PCR using primers derived from Masu et al., 1991, see above. The oligonucleotide with the sequence

5’-TATCTTGAGTGGAGTGACATAG-3 ’

(corresponding to nt 1753 to 1774 of the Masu sequence) is used as a sense initiator. The antisense initiator has the sequence

5’-ACTGCGGACGTTCCTCTCAGG-3 ’

corresponding to nt 2524 to 2544 of the Masu sequence. The C-terminal end of splice variants 1a, 1b and 1c is cleaved by PCR using primers derived from Masu et al., 1991, Tanabe et al., 1992, see above, and Pin et al., 1992 (Proc. Natl. Acad. Sci, USA, 89, 10331-10335 (1992)), respectively. The oligonucleotide that has the sequence

5’-AAACCTGAGAGGAACGTCCGCAG-3 ’

(corresponding to nt 2521 to nt 2543 of the Masu sequence) is used as a sense initiator. The oligonucleotides that have the sequences

5’-CTACAGGGTGGAAGAGCTTTGCTT-3 ’corresponding to nt 3577 to 3600 of the Masu sequence,

5’-TCAAAGCTGCGCATGTGCCGACGG-3 ’corresponding to nt 2698 to 2721 of the Tanabe sequence, and

5’-TCAATAGACAGTGTTTTGGCGGTC-3 ’corresponding to nt 2671 to 2694 of the Pin sequence are used as antisense primers for hmGluR1a, 1b and 1c, respectively.

The PCR fragment is cloned into pBluescript II and completely sequenced.

(iii) A chimeric cDNA fragment in which the i2 loop of hmGluR7a or hmGluR7b (nt 2035 to 2106 of SEQ ID 11 and 13, respectively) is replaced with the corresponding sequences of hmGluR1 is generated by PCR (as described in Pin et al., 1994, see above). The fragment is digested with Smal and Bglll that cut into unique restriction sites that flank the i2 loop. The chimeric SmaI / BglII fragment is exchanged for the SmaI / BglII fragments of pBluescript-Not-mGluR7.

(iv)

Additional replacement of the C-terminal domain of hmGluR7b or hmGluR7a with the corresponding sequences of the aforementioned hmGluR1 splicing variants is achieved through the use of the unique Bglll and SacII restriction sites that flank the C-terminal end of hmGluR7.

(v)

 The resulting chimeric cDNAs for hmGluR7 / hmGluR1 sequence and digest with Eagl, thus releasing the complete cDNAs from pBluescript-Not. For stable expression in CHO cells, the chimeric cDNAs are cloned into the unique NotI site of the mammalian expression vector pCMV-T7-2.

Example 7: Generation and application of anti-hmGluR antibodies

The peptides corresponding to the deduced amino acid sequences of the C-terminal of hmGluR4 and hmGluR7 are synthesized and coupled to ovalbumin or Tentagel. Polyclonal antisera rise in rabbits. Antibodies specific for human mGluR are purified from the antisera by means of immunoaffinity chromatography on peptide columns. Antibodies specific for hmGluR are characterized by ELISA and immunoblotting with glutathione-S-transferase / hmGluR fusion proteins (produced in E. coli) or in human brain extracts. The antibodies specific for hmGluR4 and hmGluR7, respectively, are used to detect hmGluR receptors in transfected cells and to analyze the cell and subcellular expression pattern of hmGluR receptor proteins in tissue sections of human brain material. The antibodies are raised against different specific hmGluR peptides consisting of 20 amino acids and the fusion proteins expressed in E. coli. Peptides are synthesized by solid phase synthesis, coupled to barnacle hemocyanin (KLH) or ovalbumin with glutaraldehyde. PCR fragments containing the complete putative intracellular C-terminal fragment of hmGluR are cloned as BamHI / EcoRI fragments into the E. coli pGEX-2T expression plasmid (Guan and Dixon, Analytical Biochemistry 192, 262-267 (1991 )) generating the glutathione-S-transferase (GST) / hmGluR fusion genes. E. coli DH5a cells (Gibco-BRL) carrying the expression plasmids with GST / hmGluR fusion genes at 37˚C in LB / 100 mg / ml ampicillin medium are grown overnight. The cultures are diluted 1:30 in LB and developed for 2 h at 30˚C. The expression of fusion proteins is induced by treatment with 0.1 mM isopropyl-b-D-thiogalactopyranoside for 3 h at 30 ° C. The cells are harvested by centrifugation at 5,000 x g. The fusion protein is isolated using glutathione affinity chromatography.

Deposit Information

The following plasmids were deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1 b, D-38124 Braunschweig on September 13, 1993:

Plasmid cmR1; Accession No. DSM 8549

CmR2 plasmid; DSM Access No. 8550

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Claims (28)

one.

 A purified human metabotropic glutamate (hmGluR) receptor comprising

a polypeptide having the amino acid sequence set forth in SEQ ID NO: 16.

2.

 A receptor according to claim 1 which is a subtype of hmGluR6 comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO: 16.

3.

 A purified human glutamate metabotropic receptor that is encoded by a nucleotide sequence capable of hybridizing under conditions of high stringency to SEQ ID NO: 1 or 15 and whose polypeptide product shows under a standard condition in the cAMP radioimmunoassay that AP-4 It has an agonistic effect at a concentration of less than 1 mM.

Four.

 A process for the preparation of a receptor according to claim 1, 2, or 3 comprising in vitro or in vivo multiplication of a suitable host cell transformed with a hybrid vector comprising an expression cassette that includes a promoter and a DNA encoding such a receptor whose DNA is controlled by said promoter.

5.

 The use of a receptor according to claim 1, 2, or 3 for the screening of a compound that modulates the activity of said receptor.

6.

An isolated nucleic acid comprising a nucleic acid encoding a receptor according to claim 1, 2, or 3.

7.

 A nucleic acid according to claim 6 which is a DNA.

8.

 A DNA according to claim 7 selected from the group consisting of the DNAs having the nucleotide sequence set forth in SEQ ID Nos. 1 or 15, respectively.

9.

 A process for the preparation of a nucleic acid according to claim 7 comprising chemical synthesis, recombinant DNA technology or polymerase chain reaction (PCR).

10.

 A DNA according to claim 7 which is a hybrid vector.

eleven.

 A host cell comprising a DNA of claim 10.

12.

 A eukaryotic host cell expressing the DNA of claim 10.

13.

 A host cell according to claims 11 or 12 which is a mammalian cell.

14.

 The use of a host cell according to claim 11 for the screening of a compound that modulates the activity of a receptor according to claim 1, 2, or 3.

fifteen.

 A process for the preparation of a host cell according to claim 11 comprising the transfection or transformation of the host cell with the hybrid vector of claim 11.

16.

 Purified mRNA complementary to the DNA according to claim 8.

17.

 A method for the identification of DNA encoding a subtype of hmGluR according to claim 1, 2, or 3 which comprises contacting human DNA with a probe, and identifying the DNA (s) that hybridize with said probe, wherein said probe is a nucleic acid probe comprising at least 14 contiguous nucleic acid bases according to claim 6 or 7, or the complement thereof and specifically binding to any nucleic acid of claim 6 or 7 , or the complement of it, under conditions of high rigor ".

18.

 A method for the identification of compounds that bind to a subtype of hmGluR comprising the use of a receptor protein according to claim 1, 2, or 3 in a competitive binding assay.

19.

 An assay for the identification of compounds that modulate the activity of a subtype of hmGluR according to claim 1, 2, or 3 comprising

-

 contacting the cells of claim 12 with at least one compound or signal whose ability to modulate the activity of said receptor subtype is sought, and subsequently

-

 analyze cells for a difference in the functional response that can be attributed to said receptor.

20. An assay according to claim 19 comprising contacting the cells according to claim 12 with at least one compound or signal whose ability to modulate the activity of the second messenger of a receptor subtype of the invention is sought, and subsequently

-

 control said cells by a change in the level of a second particular messenger.

twenty-one.

 A method for detecting a glutamate agonist or an allosteric modulator of a subtype of hmGluR according to claim 1, 2, or 3 having agonistic activity comprising the steps of (a) exposing a compound to a subtype of hmGluR according to with claim 1, 2, or 3 coupled with a response route, under conditions and for a time sufficient to allow interaction of the compound with the receptor and an associated response through the route, and (b) detect an increase or a decrease in the stimulation of the response route resulting from the interaction of the compound with the subtype of hmGluR, in relation to the absence of the compound analyzed and from there determine the presence of an agonist or an allosteric modulator that has agonist activity .

22

 A method for identifying a glutamate antagonist or an allosteric modulator of a subtype of hmGluR according to claim 1, 2, or 3 having antagonistic activity, said method comprising the steps of

(a) exposing a compound in the presence of a known glutamate agonist to a subtype of hmGluR according to claim 1, 2, or 3 to a response route, under conditions and for a time sufficient to allow the agonist to interact with the receptor and an associated response through the route, and (b) detect an inhibition of the stimulation of the response route by the agonist resulting from the interaction of the compound with the hmGluR subtype, relative to the stimulation of the response route induced by the glutamate agonist alone, and from there determine the presence of a glutamate antagonist or an allosteric modulator that has antagonistic activity.

2. 3.

 An antibody that specifically binds to a protein of claim 1, 2, or 3.

24.

 An antibody according to claim 23 which is a polyclonal antibody.

25.

 An antibody according to claim 23 which is a monoclonal antibody.

26.

 A method for modulating the activity and transduction of the signal of a subtype of hmGluR according to claim 1, 2, or 3 comprising contacting said receptor with an antibody of claim 24.

27.

 A receptor according to claim 1, 2, or 3 that can be obtained by means of recombinant DNA technology.

28.

 A fusion protein comprising a receptor according to claim 1, 2, or 3.