PT1199359E - Human metabotropic glutamate receptor subtypes ( hmr6) and related dna compounds - Google Patents

Description

(HMR4, HMR6, HMR7) and RELATED DNA COMPOUNDS " METABOTROPIC HUMAN GLUTAMATE RECEPTOR (HMR4, HMR6, HMR7) < / RTI > The present invention relates to human metabotropic glutamate receptor (hmGluR) proteins, isolated nucleic acids encoding them, host cells producing proteins of the invention, methods for the preparation of such proteins, nucleic acids and host cells, and their uses. 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 (guanine nucleotide binding protein) which upon binding of a glutamatergic ligand can transduce an extracellular signal through an intracellular secondary messenger system such as calcium ions , a cyclic nucleotide, diacylglycerol and inositol 1,4,5-triphosphate in a physiological response. Possessing seven putative transmembrane-spanning segments, preceded by a large extracellular amino-terminal domain and followed by a large carboxy-terminal domain, metabotropic glutamate receptors are characterized by a common structure. Based on the degree of sequence identity at the 2 ΡΕ1199359 amino acid level, the mGluR class can be divided into different subfamilies comprising individual receptor subtypes (Nakanishi, Science 258, 597-603 (1992)). Each mGluR subtype is encoded by a single gene. With respect to the homology of a single subtype mGluR subtype with another subtype of a different subfamily, the amino acid sequences are less than about 50% identical. In a subfamily, the degree of sequence identity is generally less than about 70%. Thus, a particular subtype may be characterized by its amino acid sequence homology to another mGluR subtype, especially a subtype of the same mammalian species. In addition, a particular subtype may be characterized by its region and tissue distribution, its cell and subcellular expression pattern or by its distinct physiological profile, e.g., by its electrophysiological and pharmacological properties.

Since the amino acid L-glutamate is the major neurotransmitter of excitation, it is assumed that glutamatergic systems play an important role and many neuronal processes, including synaptic transmission of rapid excitation, regulation of neurotransmitter releases, long-term potentiation , learning and memory, synaptic development plasticity, hypoxic-ischemic injury and neuronal cell death, epi-leptiform seizures, as well as the pathogenesis of various neurodegenerative disorders. To date, no information on human metabotropic glutamate receptor 3 (hmGluR) subtypes, e.g., on their amino acid sequence or tissue distribution, is available. This lack of knowledge particularly hinders research for human therapeutic agents capable of specifically influencing any disorder attributable to a defect in the glutamatergic system. In light of the physiological potential and pathological significance of metabotropic glutamate receptors, there is a need for human receptor subtypes and cells that produce these subtypes in amounts sufficient to elucidate the electrophysiological and pharmacological properties of these proteins. For example, drug screening assays require purified human receptor proteins in an active form, which has not yet been obtained. It is an object of the present invention to meet this need, namely to provide distinct subtypes of hmGluR, nucleic acids encoding them and host cells which produce such subtypes. In particular, the present invention discloses the hmGluR subfamily which comprises the subtype designated hmGluR4, and the individual proteins of said subfamily. In the following, said subfamily will be referred to as the hmGluR4 subfamily. In contrast to other hmGluR subtypes, members of this subfamily are potently activated by L-2-amino-4-phosphoutyric acid (AP4) and, when expressed in eg Chinese hamster ovary (CHO) cells or baby hamster kidney cells (BHK), coupled negatively to the adenylate cyclase via G protein. Using a system comprising a recombinant hmGluR subtype 4 of the invention in the screening for hmGluR-reactive drugs offers (among others) the possibilities of reaching a higher number of receptors by cell yielding higher reagent yield and a higher ratio of signal to noise in assays, as well as increased specificity of the receptor subtype (potentially resulting in greater biological and disease specificity). The present invention further discloses a hmGluR subtype characterized in that its amino acid sequence is more than about 65% identical to the sequence of the hmGluR4 subtype having the amino acid sequence set forth in SEQ ID NO: 2.

According to the invention, the expression " hmGluR subtype " refers to a purified protein belonging to the class of G protein-coupled receptors and which upon binding of a glutamatergic ligand transduces an extracellular signal through an intracellular secondary messenger system. In that case, a subtype of the invention is characterized by modifying 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 ion channel or other receptor. It is believed that a receptor subtype of the invention is encoded by a distinct non-coding gene. 5 ΡΕ1199359 WO9210583 discloses the identification, isolation and purification of metabotropic glutamate receptors coupled to rat G protein and antibodies against them. Nakajima et al. (1993) JBC, 268, 11868-11873 discloses the cloning and molecular characterization of a metabotropic glutamate receptor from a rat retina cDNA library. another subtype of the metabotropic glutamate receptor. A particular subtype of the invention may be characterized by its distinct psychological profile, preferably by its transduction signal and pharmacological properties. Pharmacological properties are e.g. selectivity for agonist and antagonist responses.

As defined herein, a glutamatergic ligand is eg, L-glutamate or other compound which interacts with, and binds particularly to, a glutamate type hmGluR subtype, such as ACPD (IS, 3R-1-aminocyclopentane- 3-dicarboxylic acid), an ACPD type ligand, eg QUIS (quisqualate), AP 4, and the like. Other ligands, e.g. (R, S) -α-methylcarboxyphenylglycine (MCPG) or α-methyl-L-AP4, may interact with a receptor of the invention so that glutamatergic ligand binding is prevented.

As used hereinbefore or hereinafter, the terms " purified " or " isolated " are intended to refer to a molecule of the invention in an enriched or pure form which can be obtained from a natural source or by genetic engineering. The purified proteins, DNAs and RNAs of the invention may be useful in ways that proteins, DNAs and RNAs as naturally occurring are not, such as the identification of compounds that selectively modulate the expression or activity of a hmGluR of 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. Purified hmGluR includes the purified hmGluR of the invention in a recombinant cell culture. The enriched form of a subtype of the invention relates to a preparation containing said subtype at a higher concentration than naturally, e.g., a cell membrane fraction comprising said subtype. If said subtype is in a pure form it is substantially free of other macro-molecules, particularly of naturally occurring protein contaminations. If desired, the subtype of the invention may be solubilized. One preferred subtype of the invention of purified hmGluR is a recombinant protein. Preferably, the subtype of the invention is in an active state, meaning that it has both ligand binding and signal transduction activities. The receptor activity is measured according to methods known in the art, e.g. using a binding assay or a functional assay, e.g. an assay as described below.

The hmGluR subtypes of the hmGluR4 7 ΡΕ1199359 subfamily include the hmGluR4, hmGluR7 and hmGluR6 subtypes. Described herein is the hmGluR4 subtype protein, which has the amino acid sequence shown in SEQ ID NO: 2; A hmGluR7-like protein comprising a polypeptide selected from the group consisting of polypeptides having the amino acid sequences set forth in SEQ ID NOs: 4, 6, 8 and 10, respectively. The hmGluR7 subtypes having the amino acid sequences are shown in SEQ ID NOs: 12 and 14, respectively. The hmGluR6-like protein according to the invention comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 16. Variants of receptor subtypes are also disclosed herein. For example, a variant of a hmGluR subtype is a functional or immunological equivalent of said subtype. A functional equivalent is a protein, particularly a human protein, which has a physiological profile essentially identical to the characteristic profile of said particular subtype. The physiological profile in vitro and in vivo includes receptor effector function, electrophysiological and pharmacological properties, e.g. selective interaction with agonists or antagonists. Exemplary functional equivalents may be excision variants encoded by mRNA created by alternative excision of a primary transcript, amino acid mutants, and glycosylation variants. An immunological equivalent of a particular hmGluR subtype is a protein or peptide capable of generating specific ΡΕ1199359 antibodies to said subtype. Portions of the extracellular domain of the receptor, e.g. peptides consisting of at least 6 to 8 amino acids, particularly 20 amino acids, are considered particularly useful immunological equivalents.

Other variants which are included are membrane-bound and soluble fragments and covalent or aggregative conjugates with other chemical units, these variants having one or more receptor functions, such as ligand binding or signal transduction. Exemplary fragments of hmGluR subtypes are the polypeptides having the amino acid sequences shown in SEQ ID NOs: 4, 6, 8, 10 and 16, respectively. The fragment of the invention has the amino acid sequence of SEQ ID NO: 16 and can be obtained from a natural source, either through chemical synthesis or by recombinant techniques. Because of its ability to compete with the endogenous counterpart of a hmGluR subtype, as described herein or its endogenous ligand, fragments, or derivatives thereof, which comprise the ligand binding domain are contemplated as therapeutic agents.

Covalent derivatives include, for example, aliphatic esters or amides of a carboxyl group of the receptor, O-acyl derivatives of the residues containing the hydroxyl group and the N-acyl derivatives which contain the amino group. Such derivatives may be prepared by attaching functionalities to reactive groups which are found in the side chains and the N- and C-terminal of the receptor protein. The protein of the invention may also be labeled with a detectable group, for example labeled with radioactivity, covalently attached to rare earth chelates or conjugated to a fluorescent moiety.

Other derivatives are covalent conjugates of a protein of the invention with another protein or peptide (fusion proteins). Examples are fusion proteins which comprise different portions of different glutamate receptors. Such fusion proteins may be useful for altering G protein coupling and / or improving the sensitivity of a functional assay. For example, in such fusion proteins or chimeric receptors, the intracellular domains of one subtype of the invention may be substituted with the corresponding domains of another mGluR subtype, particularly another hmGluR subtype, e.g. a hmGLuR subtype 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, e.g. mGluR1 (Masu et al., Nature 349, 760-765) or mGluR5. An intracellular domain suitable for such a switch, e.g., the second anti-cellular, also referred to as i2 (Pin et al., EMBO J. 13, 342-348 (1994)). Thus, it is possible to analyze the interaction of a test compound with a ligand binding domain of a receptor of the invention using an assay for the calcium ions. The chimeric receptor according to the invention may be synthesized by recombinant techniques or agents known in the art to be suitable for the crosslinking of proteins.

Aggregating derivatives are e.g., cell membrane adsorption complexes.

In another embodiment, the present invention relates to a composition of matter comprising a hmGluR6 subtype of the invention which comprises a polypeptide having the amino acid sequence set forth in SEQ ID

No: 16. The protein of the invention is useful e.g. as an immuno-gen in drug screening assays as a reagent for immunoassays and in purification methods such as for affinity purification of a binding attachment. The protein of the invention is obtained from a natural source, e.g. by isolation from brain tissue, by chemical synthesis or by recombinant techniques. The invention further provides a method for preparing a hmGluR6 subtype of the invention characterized in that suitable host cells which produce a subtype of the recipient of the invention are multiplied in vitro or in vivo. Preferably, the host cells are transformed (transfected) with a hybrid vector which comprises an expression cassette comprising a promoter and a DNA sequence encoding said subtype whose DNA is controlled by said promoter. Subsequently, the hmGluR subtype of the invention can be recovered. The recovery comprises e.g. isolating the subtype of the invention from the host cells or isolating the host cells comprising subtype, e.g., from the culture broth. A method for the preparation of a functionally active receptor is particularly preferred.

The HmGluR muteins can be produced from a DNA encoding a hmGluR6-like protein of the invention whose DNA has been subjected to in vitro amutagenesis which has resulted, e.g. in an addition, exchange and / or deletion of one or more amino acids. For example, substitution, deletion and insertion variants of a hmGluR subtype of the invention are prepared by recombinant and rastrader methods for immunological cross-reactivity with the native forms of hmGluR. The protein of the invention may also be derived in vitro according to conventional methods known in the art. Suitable host cells include eukaryotic cells, e.g., animal cells, plant cells and fungi, and prokaryotic cells, such as gram-positive and gram-negative bacteria, e.g. E. coli. Preferred eukaryotic host cells are those of amphibian or mammalian origin.

As used herein, in vitro means ex vivo, thereby including e.g. cell culture and tissue culture conditions.

This invention further covers an isolated nucleic acid (DNA, RNA) comprising a purified, preferably recombinant, nucleic acid (DNA, RNA) encoding a hmGluR6 type of the invention, or a fragment thereof. In addition to being useful for the production of the abovementioned recombinant hmGluR proteins, these nucleic acids are useful as probes, thereby allowing those skilled in the art to readily identify and / or isolate the nucleic acid encoding a hmGluR protein of the invention. The nucleic acid may be labeled or unlabelled with a detectable moiety. In addition, the nucleic acid according to the invention is useful eg in a method for determining the presence of hmGluR, said method comprising hybridizing the DNA (or RNA) which encodes (or is complementary to) hmGluR to test a sample of nucleic acid and to determine the presence of hmGluR. The nucleic acid of the invention encoding a purified hmGluR6-type protein includes nucleic acid which is free of at least one contaminant nucleic acid with which it is normally associated in the natural source of hmGluR nucleic acid. The purified nucleic acid is therefore present in a form or structure which is different from that found in nature. However, purified hmGluR nucleic acid encompasses the hmGluR nucleic acid in cells normally expressing hmGluR, wherein the nucleic acid is in a chromosomal location different from that of the natural cells or is otherwise flanked by a DNA sequence other than that of found in nature.

In particular, the invention provides a purified or isolated DNA molecule encoding a hmGluR6 subtype of the invention, or a fragment thereof. By definition, such DNA comprises a coding single-stranded DNA, a double-stranded DNA consisting of said coding DNA and a DNA complementary thereto, or its own complementary (single-stranded) DNA. A DNA encoding the above preferred hmGluR subtypes, or a fragment thereof, is preferred. In addition, the invention relates to a DNA which comprises such DNA. Also disclosed herein is a DNA encoding a hmGluR4 subtype or a portion thereof, particularly a DNA encoding the hmGluR4 subtype having the amino acid sequence set forth in SEQ ID NO: 2, eg the DNA with the nucleotide sequence shown in SEQ ID NO: 1. An exemplary DNA fragment encoding a portion of hmGluR4 is the cmR20 cDNA portion encoding hmGluR4, as described in the Examples. Also disclosed herein is a DNA encoding a hmGluR7 subtype, particularly a DNA encoding any of the hmGluR7 subtypes having the amino acid sequences set forth in SEQ ID NOs: 12 and 14, respectively, eg the DNAs having the nucleotide sequences -otides shown in SEQ ID NOs: 11 and 13, respectively. Also disclosed is a DNA fragment encoding a portion of a hmGluR7 subtype, particularly the hmGluR7 subtypes identified above as being preferred. Exemplary fragments of hmGluR7 DNA include the cmR2, cmR3, cmR5 and CR7PCR1 cDNA moieties encoding hmGluR7 as described in the Examples, or a DNA fragment encoding substantially the same amino acid sequence as is encoded by the portion of the cmR2 plasmid encoding hmGluR7 , filed on DSM on September 13, 1993, under the accession number DSM 8550. These DNAs encode portions of putative hmGluR7 subtype variant variants described herein.

In particular, the invention provides a DNA encoding a hmGluR6 subtype or a portion thereof, particularly a DNA encoding the hmGluR6 subtype moiety, the amino acid sequence shown in SEQ ID NO: 16, or a DNA encoding substantially the same amino acid sequence as that encoded by that portion of the plasmid cmR1 encoding hmGluR6, deposited with DSM on September 13, 1993, under accession number DSM 8549. An exemplary DNA sequence is shown in SEQ ID NO: 15. 15 ΡΕ1199359

The nucleic acid sequences provided herein may be employed to identify DNAs encoding other hmGluR subtypes. For example, the nucleic acid sequences of the invention may be used to identify DNAs encoding other hmGluR subtypes belonging to the subfamily comprising hmGluR4. A method for identifying DNA comprises contacting human DNA with a nucleic acid probe described above and identify the DNA (s) that hybridize to that probe. Exemplary nucleic acids of the invention may be alternatively characterized as the nucleic acids encoding a hmGluR subtype of the invention and hybridize under the high stringency conditions 1 to a DNA sequence shown in SEQ ID NOs. 1 or 15, or a portion (fragment) selected from said DNA sequence. For example, fragments selected useful for hybridization are those portions that encode the said DNAs proteins. The stringency of hybridization refers to the conditions under which the polynucleic acid hybrids are stable. These conditions are obvious to those with normal field experience. As is well known to those skilled in the art, the stability of the hybrids is reflected at the melt temperature (Tm) of the hybrid which decreases by approximately 1 to 1.5 ° C and a 1% decrease in sequence homology. In general, the stability of a 16 ΡΕ1199359 hybrid is a function of the concentration of sodium ions and the temperature. Typically, the hybridization reaction is performed under the highest stringency conditions, followed by washings of varying stringency.

As used herein, high stringency refers to conditions that allow the hybridization of only those nucleic acid sequences that form stable hybrids in 1 MNa + at 65-68 ° C. High stringency conditions may be provided, for example, by hybridization in an aqueous solution containing 6x SSC, 5x Denhardfs, 1% SDS (sodium dodecylsulfate), 0.1 Na + pyrophosphate and 0.1 mg / ml DNA of denatured salmon sperm as a non-specific competitor. After hybridization, a high stringency wash can be performed in several steps, with a final wash (about 30 min) at the hybridization temperature in 0.2-0.1% SSC, 0.1% SDS. Moderate stringency refers to conditions equivalent to hybridization in the solution described above, but at about 60-62Â ° C. In that case, the final wash is performed at the hybridization temperature in 1x SSC, 0.1% SDS. The low stringency refers to conditions equivalent to hybridization in the solution described above at about 50-52Â ° C. In that case, the final wash is performed at the hybridization temperature in 2x SSC, 0.1% SDS.

It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g., formamide-based buffers, and temperatures. The Denhart and SSC solution are well known to those skilled in the art, as are other suitable hybridisation buffers (see, eg, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA, or Ausubel, FM, et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, USA). Optimum conditions of hybridization have to be determined empirically, insofar as the length and GC content of the probe also play a relevant role.

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

For example, a DNA of the invention can be obtained by chemical synthesis, by recombinant DNA technology or by the polymerase chain reaction (PCR). Preparation by recombinant DNA technology may involve screening a suitable cDNA or genomic library. A suitable method for preparing a DNA of the invention comprises the synthesis of various oligonucleotides, their amplification by PCR methods, and their excision to produce the desired 18 ΡΕ1199359 DNA sequence. Suitable libraries are available commercially, e.g. the libraries employed in the Examples, or may be prepared from neural or neuronal tissue samples, e.g., hippocampal and cerebellum tissue, cell lines and the like.

For the individual hmGluR subtypes (and excision variants) of the invention, the expression pattern in neural or neuronal tissue may vary. Thus, in order to isolate the cDNA encoding a particular subtype (or excision variant), it is advantageous to screen libraries prepared from different suitable tissues or cells. As a screening probe, a DNA or RNA which substantially comprises the entire coding region of a hmGluR subtype of the invention, or a suitable oligonucleotide probe based on said DNA, may be employed. A suitable oligonucleotide probe (for screening involving hybridization) is a double stranded DNA or RNA that has a nucleotide sequence that includes at least 14 contiguous bases that are equal to (or complementary to) any 14 or more contiguous bases shown in any of SEQ ID NOs: 1, or 15. The probe may be labeled with a suitable chemical moiety for rapid detection. The nucleic acid sequences selected as probes must be of sufficient length and must be sufficiently unambiguous so that false positive results are minimized.

Preferred regions from which probes may be constructed include the 5 'and / or 3' coding sequences, sequences provided as encoding ligand binding sites, and the like. For example, both the full length cDNA clones disclosed herein and fragments thereof can be used as probes. Preferably, the nucleic acid probes of the invention are labeled with labeling means suitable for rapid detection upon hybridization. For example, a suitable labeling medium is a radioactive label. The preferred method of marking a DNA fragment is through incorporation of 32 P-labeled α-dATP with the Klenow fragment of DNA polymerase in a random primer reaction, as is well known in the art. Oligonucleotides are typically labeled at the 32 'α-ATP and polynucleotide kinase endpoints. However, other methods (e.g., non-radioactive) may also be used to label the fragment or oligonucleotide, including e.g. enzyme labeling and biotinylation.

After screening the library, e.g. with a portion of DNA including substantially the entire sequence encoding hmGluR or a suitable oligonucleotide based on a portion of said DNA, positive clones are identified through the detection of a hybridization signal; the identified clones are characterized by restriction enzyme mapping and / or DNA sequence analysis, and then examined, eg by comparison with the sequences shown herein, to ensure that DNA encoding a complete hmGluR is included (ie, if ΡΕ1199359 they include translation initiation and termination codons). If the selected clones are incomplete, they can be used to re-screen the same or a different library to obtain overlapping clones. If the library is genomic, then the overlapping clones may include introns and exons. If the library was a cDNA library, then the overlapping clones will include an open reading frame. In both cases, complete clones can be identified by comparison to the DNA sequences and deduced amino acid sequences provided herein.

Furthermore, as far as detecting any abnormality of an endogenous hmGluR6 subtype of the invention, genetic screening can be performed using the nucleotide sequences of the invention as hybridization probes. Also, based on the nucleic acid sequences provided herein, therapeutic agents of the antisense type can be designed.

It is contemplated that the nucleic acid of the invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a number of nucleotides, and any combination thereof. Such modified sequences may be used to produce a mutant subtype of hmGluR6 that differ in receptor subtypes found in nature. Mutagenesis can be predetermined (site-specific) or randomized. A mutation that is not a silent mutation should not place the sequences outside the deletion gratings and preferably will not create complementary regions that can hybridize to produce secondary mRNA structures such as loops or hooks. The cDNA or genomic DNA encoding native or mutant hmGluR6 of the invention may be incorporated into the vectors for further manipulation. In addition, the invention relates to a recombinant DNA which is a hybrid vector comprising at least one of the DNAs mentioned above.

The hybrid vectors of the invention comprise a replication origin or autonomous replication sequence, one or more dominant marker sequences, and optionally, expression control sequences, signal sequences and additional restriction sites.

Preferably, the hybrid vector of the invention comprises an inserted nucleic acid inserted operably 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 hmGluR6 subtype of the invention, i.e. to produce usable amounts of the nucleic acid (cloning vectors). The other function is to provide the replication and expression of the genetic constructs in a suitable host, either by maintaining an extracromosomal element or by integrating into the host chromosome (expression vectors). A cloning vector comprises the DNAs as described above, an origin of replication or an autonomous replication sequence, select marker sequences, and optionally, signal sequences and additional restriction sites. An expression vector further comprises expression control sequences essential for the 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, recombinant viruses or another vector which, upon introduction into a suitable host cell, results in the expression of the cloned DNA. Suitable expression vectors are 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 may 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 if it is not capable of replicating independently of the host cell chromosome. The DNA can also be amplified by insertion into the host genome. Contained, the recovery of the genomic DNA encoding hmGluR is more complex than the 23 ΡΕ1199359 of the exogenously replicated vector because restriction enzyme digestion is required to excise the hmGluR DNA. The DNA can be amplified by PCR and directly transfected into the host cells without any replication component.

Advantageously, the expression and cloning vector contains the selection gene also referred to as a selection marker. This gene encodes a protein necessary for the survival or culturing of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the deletion gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, auxotrophic complement deficiencies, or supply of critical nutrients not available from the complex medium.

Since the amplification of the vectors is conveniently performed in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript vector or a pUC plasmid.

Suitable selection markers for mammalian cells are those that allow the identification of cells competent to incorporate hmGluR nucleic acid, such as dihydrofolate reductase (DHFR, resistance to methotrexate), thymidine kinase, or genes that confer resistance G418 or hygromycin. Mammalian cell transfectants are placed under the selection pressure to which they are only uniquely adapted to survive those transfectants that have incorporated and expressed the tag.

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

Suitable promoters for use with prokaryotic hosts include, for example, the β-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and 25 ΡΕ1199359 hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker to operably link the DNA encoding hmGluR, using linkers or adapters to provide at any necessary restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgamo sequence operably linked to the DNA encoding hmGluR. Transcription of the HmGluR gene from vectors in mammalian host cells can be controlled by promoters compatible with host cell systems, e.g. promoters derived from the genomes of the viruses. Plasmids for expression of a hmGluR6 subtype of the invention in eukaryotic host cells, particularly mammalian cells, are eg vectors containing the cytomegalovirus (CMV) promoter, vectors containing the RSV promoter and vectors containing the SV40 promoter and vectors contain the promoter of MMTV LTR. Depending on the nature of their regulation, the promoters may be constitutive or regulatable by experimental conditions. Transcription of a DNA encoding a hmGluR6 subtype according to the invention by higher eukaryotes can be enhanced by inserting an enhancer sequence into the vector.

The various DNA segments of the vector DNA are operably linked, i.e. they are contiguous and placed in functional relationship to each other. The construction of vectors according to the invention employs conventional binding techniques. The isolated plasmids or DNA fragments are cleaved, tailor-made, and religated in the desired form to create the required plasmids. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a manner known in the art. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyzes to evaluate the expression and function of hmGluR are known to those skilled in the art. The presence, amplification and / or expression of the gene may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate mRNA transcription, drop transfer (DNA or RNA analysis), hybridization 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 readily understand how these methods can be modified, if desired. The invention further provides host cells capable of producing a hmGluR6 subtype of the invention and including heterologous (foreign) DNA encoding said subtype. 27 ΡΕ1199359

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

Suitable prokaryotes including eubacteria, such as Gram-negative or Gram-positive organisms, such as E. coli, e.g. E. coli strains K-12, DH5Î ± and HB 101, or Bacilli. Other suitable host cells for vectors encoding hmGluR include eukaryotic microbes such as filamentous fungi or yeast, e.g. Saccharomyces cerevisiae. Higher eukaryotic cells include insect, amphibian and vertebrate cells, particularly mammalian cells, e.g. neuroblastoma cell lines or fibroblast-derived cell lines. Examples of preferred mammalian cell lines are e.g. 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 vertebrate cells into culture (tissue culture) has become a routine process. The host cells referred to in this application comprise in vitro cultured cells as well as cells which are in an animal host. 28 ΡΕ1199359

Host cells suitable for expression of an hmGluR6-like recombinant active protein of the invention advantageously express endogenous or recombinant G proteins. Preferred are cells which produce little, if any, endogenous metabo-tropic glutamate receptor. The DNA may be stably incorporated into the cells or may be transiently expressed according to standard methods.

Stably transfected mammalian cells can be prepared by transfecting the cells with an expression vector having the selection marker gene, and culturing the transfected cells without conditions selective for the cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor the transfection efficiency.

To produce such transiently or stably transfected cells, the cells must be transfected with a sufficient amount of hmGluR-encoding nucleic acid to form the hmGluR6-like protein of the invention. The precise amounts of DNA encoding the hmGluR6-like protein of the invention can be determined empirically and optimized for a particular cell and assay. The DNA of the invention may also be expressed in 29 ΡΕ1199359 non-human transgenic animals, particularly transgenic warm-blooded 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 comprising a DNA of the invention encoding the hmGluR6-like protein coupled with appropriately positioned expression control sequences is introduced into the pronuclei of the fertilized eggs. The introduction can be achieved, e.g. by microinjection. Integration of the injected DNA is detected, e.g. by DNA transfer analysis from suitable tissue samples. It is preferred that the introduced DNA be incorporated into the germ line of the animal so that it is passed to the offspring of the animal. Preferably, a transgenic animal is developed by targeting a mutation to disrupt a hmGluR sequence. Such an animal is useful e.g. to study the role of a metabotropic receptor metabolism.

In addition, an animal with the deactivated gene can be developed by introducing a mutation in the hmGluR sequence, thereby creating an animal that no longer expresses the functional hmGluR gene. Such an inactivated gene animal is useful e.g. to study the role of the metabotropic receptor in metabolism methods to produce mice with the inactivated gene which are known in the art. 30 ΡΕ1199359

The host cells are transfected or transformed with the above expression and cloning vectors of this invention and cultured in conventional nutrient media, modified as appropriate for induction promoters, selecting the transformants, or amplifying the genes encoding the desired sequences. The heterologous DNA may be introduced into the host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate co-precipitation technique, by electroporation or by lipo-fectin mediated. Numerous methods of transfection are known to those skilled in the art. Successful transfection is generally recognized when any indication of the operation of this vector occurs in the host cell. The transformation is achieved using standard techniques appropriate to the particular host cells used. Incorporation of the cloned DNA into a suitable expression vector, transfection of eukaryotic cells with a plasmid vector or a combination of plasmid vectors, each encoding one or more distinct genes or with linear DNA, and selection of transfected cells are well known in the art (see, eg, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press). 31 ΡΕ1199359

Transfected or transformed cells are cultured using culture media and methods known in the art, preferably under conditions, according to which the DNA encoded by hmGluR is expressed. It is known to those who are in the area a suitable medium composition, so that it can be quickly prepared. Suitable culture media are also commercially available.

Although the DNA provided herein may be expressed in any suitable host cell, eg those referred to above, preferred for expression of DNA encoding functional hmGluR are eukaryotic expression systems, particularly mammalian expression systems, including commercially available systems and other systems known to the human skilled in the art. DNA of the human mGluR6 type protein of the invention is linked to a vector, and is introduced into host cells suitable for producing transformed cell lines expressing a particular hmGluR6 subtype of the invention, or specific combinations of subtypes. The resulting cell line can then be produced in amounts sufficient for reproducible qualitative analysis and quantitative effects of a receptor agonist, antagonist or allosteric modulator. Additionally, the mRNA can be produced by in vitro transcription of a DNA encoding a subtype of the invention. This mRNA can be injected into Xenopus oocytes where the mRNA controls the synthesis of the active receptor subtype. Alternatively, the DNA encoding the subtype can be injected directly into the oocytes. Transfected mammalian cells or injected oocytes can then be employed in a drug screening assay presented below. Such drugs are useful in diseases associated with the pathogenesis of an hmGluR6 subtype of the invention. Such diseases include diseases resulting from the excessive action of glutamate, preferably mediated by hmGluRs, such as thrombosis, epilepsy and chronic neurodegenerative diseases. Particularly useful for evaluating the specific interaction of compounds with the specific hmGluR6s subtype of the invention are stably transfected cell lines expressing an hmGluR of the invention.

These host cells expressing the hmGluR6-like protein of the invention are useful for drug screening and it is a further object of the present invention to provide a method for identifying a compound or signal that modulates hmGluR activity, said method comprising exposing the cells which contain the heterologous DNA encoding the hmGluR6-like protein of the invention, wherein said cells produce functional hmGluR, for at least one compound or signal whose ability to modulate the activity of said hmGluR is determined, and thereafter monitor said cells in changes caused by such modulation. Such an assay allows the identification of allosteric agonists, antagonists and modulators of an hmGluR of the invention. 33 ΡΕ1199359

In a further aspect the invention relates to an assay for identifying compounds that modulate the activity of a hmGluR6 subtype of the invention, said assay comprising: contacting cells expressing an active hmGluR subtype of the invention and containing the DNA heterologous peptide which encodes said hmGluR6 subtype with at least one compound to be tested for its ability to modulate the activity of said receptor, and - to analyze the cells for a difference at the level of the secondary messenger or receptor activity.

In particular, the invention covers an assay for identifying compounds that modulate the activity of a hmGluR subtype of the invention, said assay comprising: contacting cells expressing the hmGluR6-like active protein of the invention and containing the heterologous DNA encoding the said hmGluR6 subtype with at least one compound to be tested as to its ability to modulate the activity of said receptor, and to monitor said cells ΡΕ1199359 for a resulting change in secondary messenger activity. The result obtained in the assay is compared with a suitable assay as a negative control.

The test methods generally need to be compared with several controls. A change in receptor activity or at the secondary messenger level is said to be induced by a test compound if that effect does not occur in the absence of the test compound. An effect of a test compound on a receptor subtype of the invention is said to be mediated by said receptor if this effect is not observed on cells that do not express the receptor.

As used herein, a compound or signal that modulates the activity of a hmGluR6-like protein of the invention relates to a compound or signal that alters the response pathway mediated by said hmGluR in a cell (as compared to the absence of said hmGluR) . A response pathway is activated by an extracellular stimulus, resulting in a change in a secondary messenger concentration or enzyme activity, or resulting in a change in the activity of the membrane bound protein, such as a receptor or ion channel. A variety of response pathways may be used, including for example, the adenylate cyclase response pathway, the calcium phospholipase C / intracellular calcium response, or coupling to a 35 ΡΕ1199359 ion channel. Assays for determining adenylate cyclase activity are well known in the art, and include e.g. the assay disclosed by Nakajima et al., J. Biol. Chem. 267, 2437-2442 (1992)).

Thus, cells expressing the hmGluR6-like protein of the invention may be employed for the identification of the compounds, particularly low molecular weight molecules capable of acting as agonists or glutamate antagonists. Low molecular weight molecules less than 1000 Daltons are preferred. In the context of the present invention, it is understood that an agonist refers to a molecule that is capable of interacting with a receptor, thereby mimicking the action of L-glutamate. In particular, a glutamate agonist is characterized by its ability to interact with an hmGluR6-like protein of the invention, and thereby increase or decrease the stimulation of a response pathway in a cell. For example, an agonist either increases or decreases a measurable parameter in the host cell such that the concentration of a secondary messenger, as with the natural ligand, increases or decreases said parameter. For example, in a suitable assay system, wherein the hmGluR6-like protein of the invention is negatively coupled to the adenylate cyclase, eg CHO or BHK cells expressing an hmGluR of the invention, such an agonist is capable of modulating the function of said hmGluR so that intracellular concentration of cAMP is decreased. 36 ΡΕ1199359

In contrast, in situations where it is desirable to lower the activity of hmGluR, the antagonizing molecules are useful. In 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 which does not stimulate a response pathway in a cell. In particular, glutamate antagonists are generally identified by their ability to interact with a hmGluR6-like protein of the invention, and thereby reduces the ability of the natural ligand to stimulate a response pathway in a cell, eg through interference with L -glutamate to a hmGluR6-like protein of the invention or inhibiting other cellular functions necessary for hmGluR activity. For example, in a suitable assay, eg an assay involving CHO or BHK cells expressing a hmGluR6 subtype type protein of the invention, a glutamate antagonist is capable of modulating the activity of an hmGluR6 type protein of the invention, so that the ability of the natural ligand to decrease the concentration of intracellular cAMP is weakened. Yet another alternative to achieve an antagonistic effect is to rely on overexpression of hmGluR antisense RNA. An agonist or antagonist that selectively acts on a protein receptor of the hmGluR6 subfamily, e.g., hmGluR6, is preferred. Particularly useful is an agonist or antagonist, which specifically modulates the activity of a particular hmGluR subtype without affecting the activity of any other subtype. 37 ΡΕ1199359

An allosteric modulator of an hmGluR6-like protein of the invention interacts with the receptor protein at a site other than 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 acting as an agonist may enhance the specific interaction between a hmGluR6-like protein of the invention and L-glutamate. If an allosteric modulator acts as an antagonist, it can e.g. interact with the receptor protein so that agonist allelication is functionally less effective.

As an in vitro assay for a glutamate agonist or antagonist it may require that a hmGluR6-like protein of the invention be produced in sufficient amounts in a functional form using recombinant DNA methods. An assay is then devised to measure a functional property of the hmGluR protein, e.g. interaction with a glutamatergic ligand. The production of a hmGluR6-like protein of the invention is envisaged as occurring in sufficient amounts if the activity of said receptor results in a measurable response.

For example, mammalian cells, eg HEK293 cells, L cells, CH0-K1 cells, LLCPK-1 cells or GH3 cells (available eg from the American Tissue Type Culture Collection) are adapted for culture in a reduced glutamate medium, preferably a free medium of 38 ΡΕ1199359 glutamate. An hmGluR expression plasmid, eg a plasmid described in the Examples, is transiently transfected into the cells, eg by calcium phosphate precipitation (Ausubel, FM, et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, USA). Cell lines that stably express an hmGluR6-like protein of the invention, eg through lipofectin mediated transfection with hmGluR expression plasmids and a plasmid comprising a selection marker gene, eg pSV2-Neo (Southern and Berg , J. Mol. Appl. Genet., 1, 327-341 (1982)), a vector plasmid encoding the G-418 resistance gene. Cells surviving the selection are isolated and cultured in the selection medium. Resistant clonal cell lines, e.g. for immunoreactivity with subtype-specific antibodies against hmGluR or by assays for functional responses of hmGluR after addition of the agonist, are analyzed. Cells producing the desired hmGluR subtype are used in a method for detecting compounds binding to a hmGluR6-like protein of the invention or in a method for identifying a glutamate agonist or antagonist.

In another embodiment, the invention provides a method for identifying binding compounds to a hmGluR6 subtype, said method comprising employing a hmGluR6 subtype of the invention in a competitive binding assay. The principle underlying a competitive binding assay is generally known in the art. 39 ΡΕ1199359

Briefly, binding assays according to the invention are performed allowing the compound to be tested for its ability to bind hmGluR to compete with a known, suitably labeled, glutamatergic ligand for the target site on the hmGluR target molecule. A suitably labeled ligand is e.g. a radiolabeled ligand, such as [3H] glutamate, or a ligand that can be detected by its optical properties, such as absorbance or fluorescence. After removing the unbound ligand and the test compound the amount of labeled ligand is measured to the hmGluR. If the amount of labeled ligand is reduced in the presence of the test compound, this compound is referred to as being bound to the target molecule. A competitive binding assay may be performed e.g. with transformed or transfected host cells expressing a hmGluR of the invention or a membranous cell fraction comprising a hmGluR6-like protein of the invention. The compound bound to the target hmGluR can modulate the functional properties of hmGluR and may therefore be identified with a glutamate agonist or antagonist in a functional assay.

Functional assays are used to detect a change in the functional activity of a hmGluR6-like protein of the invention, i.e. to detect a functional response, e.g. as a result of the interaction of the compound to be tested with said hmGluR. A functional response ΡΕ1199359 is eg a change (difference) in the concentration of a relevant secondary messenger, or a change in the activity of another membrane bound protein bound by the receptor of the invention on cells expressing a functional hmGluR of the invention (compared to a negative control). Those skilled in the art can readily identify a suitable assay to detect a change in the level of an intracellular secondary messenger indicative of the expression of an active hmGluR (functional assay). Examples include cAMP assays (see, eg, Nakajima et al., J. Biol. Chem. 267, 2437-2442 (1992), cGMP assays (see, eg Steiner et al., J. Biol. Chem., 247, 1106 (Nakajima et al., J. Biol. Chem. 267, 2437-2442 (1992)), calcium ion flow assays (Ito et al., J. Biol. Chem. Neurochem 56, 531-540 (1991)), arachidonic release assays (see, eg Felder et al., J. Biol. Chem. 264,20356-20362 (1989)), and the like.

More specifically, according to the invention a method for detecting a glutamate agonist comprises the steps of (a) exposing a compound to a hmGluR6 subtype of the invention coupled to a response path, under conditions and for a time sufficient to allow the interaction of the compound with the receptor and an associated response across the pathway, and (b) detecting an increase or decrease and stimulation of the response pathway resulting from the interaction of the compound with the hmGluR6 subtype relative to the absent compound tested and thereby determining the presence of a compound agonist of glutamate. 41 ΡΕ1199359

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 hmGluR6 subtype of the invention coupled to a response path, under conditions and for a time sufficient to allow the interaction of the agonist with the receptor and an associated response across the pathway, and (b) detecting an inhibition of agonist response pathway stimulation resulting from the interaction of the compound with the hmGluR6 subtype, in relation to the stimulation of the agonist response pathway glutamate alone and subsequently determine the presence of a glutamate antagonist. Inhibition can be detected, e.g. if the test compound competes with the glutamate agonist for the hmGluR6 subtype of the invention. Compounds that can be screened using this method are e.g. to block antibodies that bind specifically to the hmGluR6 subtype. Furthermore, such assay is useful for screening for compounds interacting with L-glutamate, e.g. soluble hmGluR fragments, which comprise part or all of the binding to the ligand domain.

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

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

In one embodiment of the present invention, the response pathway is a membrane bound adenylate cyclase pathway, and, for an agonist, the detection step comprises measuring a reduction or increase, preferably a reduction, in the production of cAMP via response of the membrane bound adenylate cyclase, relative to the production of cAMP in the establishment of the relevant control. For the purpose of the present invention, it is preferred that the reduction or increase in the production of cAMP is equivalent to or greater than the reduction or increase induced by L-glutamate applied at a concentration corresponding to its concentration of IC50. For an antagonist, the detection step comprises measuring the present antagonist of a smaller L-glutamate-induced decrease or increase in cAMP production by the membrane-bound adenylate cyclase response pathway, compared to the production of cAMP in the absence of the antagonist. Measurement of cAMP may be performed after cell destruction or a cAMP-sensitive molecular probe 43 applied to the cell, such as a fluorescent dye, which alters its properties, e.g., its fluorescent properties, upon binding of cAMP. Cyclic AMP production can be measured using methods well known in the art, including for example the methods described by Nakajima et al., Supra, or using commercially available kits, eg kits which comprise radioactively labeled cAMP, eg [125 I] cAMP or [3H] cAMP. Exemplary kits are Amersham's Scintillation Proximity Assay Kit, which measures the production of CAMP through the competition of cAMP-iodinated cAMP antibodies, or the Amersham Cyclic AMP [3H] Assay Kit.

In assay systems using cells expressing receptor subtypes that are negatively coupled to the adenylate cyclase pathway, i.e. which causes a decrease in cAMP after stimulation and an increase in cAMP upon reduction of stimulation, it is preferred to expose the cells to a compound which stimulates reversibly or irreversibly the adenylate cyclase, eg forcoprotein, or which is a phosphodiesterase inhibitor, such as isobutylmethylxanthine (IBMX) prior to addition of the (potential) agonist or receptor antagonist.

In another embodiment of the invention, the response pathway is the route of mobilization of the Ca2 + hydrolyzate PI. Such assay to determine the specific interaction of a test compound with a hmGluR6 subtype of the invention 44 ΡΕ1199359 may be functionally linked to changes in concentration of intracellular (Ca2 +) calcium ions. Various methods are known in the art for determining a change in the intracellular concentration of Ca 2+, eg a method involving a calcium ion sensitive fluorescent dye such as fura-2 (see Grynkiewisz et al., J. Biol. Chem. 260, 3440- 3450, 1985), fluo-3 or Indo-1, such as the QuinZ method of fluorine calcium described by Charest et al. (1993)), or the equine photoprotein method described by Nakajima-Shimada (Proc Natl Acad Sci USA 88, 6878-6882 (1991)). In one embodiment of the invention, the concentration of intracellular calcium ion is measured by microfluorometry in recombinant cells loaded with fluorescent dyes sensitive to calcium fluo-3 or fura-2. These measurements may be performed using cells cultured on a slide allowing the use of an inverted microscope and video imaging technologies or a fluorescence photometer to measure calcium concentrations at the single cell level. For both approaches, cells transformed with a plasmid expressing 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 preferably over the next 8 h. Microfluorometry follows conventional procedures.

The Ca2 + signals resulting from the functional interaction of the compounds with the target molecule may be transient if the compound is applied for a limited time, e.g. through an infusion system. Using the transient application several measurements can be made with the same cells that allow for internal controls and high numbers of tested compounds.

Functional coupling of a hmGluR6-like protein of the invention to the Ca2 + signaling pathway, eg in CHO cells, can be achieved by various methods: (i) coexpression of a recombinant hmGluR6-like protein of the invention and a cation channel with a recombinant voltage port, an activity to which it is functionally linked to the activity of hmGluR; (ii) expression of a hmGluR chimeric receptor, which directly stimulates the PI / Ca2 + pathway; (iii) coexpression of a recombinant hmGluR6-like protein of the invention with a recombinant Ca 2+ -permanent cAMP-dependent cation channel.

In other expression systems the functional coupling of a hmGluR to Ca2 + signaling can be achieved by transfection of a hmGluR of the invention if these cells naturally express (i) the voltage channel Ca channels, an activity of which is functionally linked to the activity of mGluRs or (ii) ion channels 46 ΡΕ1199359 dependent on Ca2 + permeable cAMP. For example, GH3 cells which naturally express the voltage port Ca channels, allow direct application of Ca 2+ assays to test the functional activity of hmGluR by co-transfection of hmGluRs.

Other cell-based screening assays may be designed e.g. by constructing cell lines in which the expression of a reporter protein, i.e. a readily assayable protein, such as β-ase, chloramphenicol acetyltransferase (CAT) or luciferase, is dependent on the function of a hmGluR6-like protein of the invention. For example, a DNA construct comprising a cAMP response element is operably linked to the luciferase encoding DNA. The resulting DNA construct comprising the enzyme DNA is stably transfected into a host cell. The host cell is then transfected with a second DNA construct containing a first DNA segment encoding a hmGluR6-like protein of the invention operably linked to additional DNA segments required for receptor expression. For example, if binding of a test compound to the hmGluR6 subtype of the invention results in elevated cAMP levels, luciferase expression is induced or decreased, depending on the promoter chosen. Luciferase is exposed to luciferin, and the photons emitted during the luciferin oxidation by luciferase is measured.

The drug screening assays provided herein will allow identification and design of receptor subtype specific compounds, particularly receptor binding ligands, eventually leading to the development of a disease specific drug. If it is designed for a very specific interaction with only a particular subtype of hmGluR6 (or a predetermined selection of hmGluR6s subtype), it is more likely that such drug will exhibit fewer undesirable side effects than a drug identified by screening with cells expressing a (unknown ) of receptor subtypes. Also, testing a single receptor subtype of the invention or specific combinations of different receptor subtypes with a variety of potential agonists or antagonists provides 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 very specific interaction with one or more receptor subtypes.

In another embodiment, the invention provides polyclonal and monoclonal antibodies raised against a hmGluR6 subtype of the invention. Such antibodies may be useful e.g. for immunoassays including immunohistochemistry, as well as for diagnostic and therapeutic applications. For example, antibodies specific for the extracellular domain, or portions thereof, of a particular subtype of hmGluR6 can be applied to block the endogenous subtype of hmGluR6. 48 ΡΕ1199359

Antibodies of the invention may be prepared according to methods well known in the art using as antigen a hmGluR6 subtype 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. The antibodies may be able to distinguish between the active or inactive form. Factors to consider in selecting fragments of the subtype as antigens (either as synthetic peptide or as fusion protein) include antigenicity, accessibility (i.e., extracellular and cytoplasmic domains) and singularity of the particular subtype.

Particularly useful are the antibodies that selectively recognize and bind to subtypes of the receptor of the above-described subfamily without binding to a sub-type 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 may be administered to a subject in need thereof using standard methods. One skilled in the art can readily determine dosage forms, treatment regimens, etc., depending on the mode of administration employed. The invention particularly relates to the specific embodiments as described in the Examples 49 and 4999359 serve to illustrate the present invention but should not be construed as a limitation thereof.

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

Example 1: cDNA encoding hmGluR4

Human mGluR4 cDNA clones are isolated from human fetal brain and human cerebellum cDNA libraries by low stringency hybridization using a radioactive labeled mouse demGluR4 probe produced by PCR from mouse brain cDNA. 1.1 Preparation of poly (A) + mouse stem-fed RNA: Adult male Sprague-Dawley rats are killed by suffocation, their pro-cephalon is removed and immediately frozen in liquid N2. Total RNA is isolated using the guanidine thiocyanate procedure (Chomczynski and Sacchi (1987), Anal. Biochem 162, 156-159). The enrichment of poly (A) + RNA is achieved by oligo (dT) -cellulose affinity chromatography 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). 50 ΡΕ1199359 1.2 Synthesis of the first cDNA strand for PCR: Poly (A) + (mRNA) RNA is reverse transcribed into DNA by Moloney Virus Reverse Transcriptase from

Murine Leukemia (M-MLV RT, BRL). Reactions of 50 Âμl are prepared as follows: 10 Âμg of mouse pro-cephalon of poly (A) + RNA in 10 Âμl of sterile H20 are heated at 70Â ° C for 10 min and then rapidly cooled in ice. Then 10 Âμl of 5x reaction buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2), 5 Âμl of 0.11 dithiothreitol, 5 Âμl dNTP mixture (10 mM of each dATP, dCTP, dGTP, dTTP, Pharmacia), 1.25 ol oligo-dTi 2 -18 (2 mg / mL, Pharmacia), 2.5 pL of RNAsin (40U / mL, Promega), 12.25 mL of H20 and 4 mL (200 U / mL) of M-MLV RT are added. The reaction is run at 37 ° C for 60 min. 1.3 PCR conditions to produce the rat mGluR4 fragment: The oligodeoxynucleotide primers used for PCR are synthesized by the phosphoramidite method. The sequences are listed in Table 1.

Conventional PCR conditions for a 100 ÂμL reaction mixture are: 30 ng of rat pro-cephalic cDNA, 50 pmol of each of the PI and P2 primers, 200 mmol of each of the four deoxynucleoside triphosphates dATP, dCTP, dGTP and dTTP, 10% DMSO PCR amplification (10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2, 50 mM KCl, 10 mM β-mercaptoethanol, 0.05%

Tween (w / v), 0.05% NP-40 (w / v)), and 0.5 U of AmpliTaq

Polymerase (Perkin Elmer Cetus). 51 ΡΕ1199359

Table 1

Pl: 5'-GTCAAGGCCTCGGGCCGGGA -3 'corresponding to 1921-1940 bp of rat mGluR4 cDNA [Tanabe et al., Neuron 8: 169-179 (1992)] P2: 5'-CTAGATGGCATGGTTGGTGTA-3' corresponding to 2788- 2808 bp of rat mGluR4 cDNA [Tanabe et al., Neuron 8: 169-179 (1992)] Amplification is performed using the following conditions: 30 sec denaturation at 93øC, 1 min 30 sec pairing at 56 ° C, and 3 min extension at 72 ° C, for a total of 40 cycles. Initial denaturation is performed for 4 min at 94 ° C. 1.4 Subcloning of the mouse mGluR4 PCR fragment: Restriction endonuclease digestions, use of modifying enzymes, vector preparation (dephosphorylation, gel purification), ligation, transformation of E. coli, and plasmid DNA preparations are performed from according to standard procedures (Sambrook, et al. (1989), supra). The PCR fragment (888 bp) obtained according to the procedure described in 1.3 is ligated into the SmaI site of the Bluescript SK + plasmid (Stratagene, La Jolla, USA). The fragment inserted into the Bluescript vector is sequenced from T7 and T3 both ends using the primers (Stratagene, La Jolla, USA). 52 ΡΕ1199359 1.5 Preparation of a radioactively labeled probe: 20-50 ng of the PCR produced from rat mGluR4 fragment are purified from the gel and 32P-labeled by random priming using a DNA Labeling Kit (Boehringer Mannheim). 1.6 cDNA library search: About 1 x 106 phages from a human fetal brain library (λΖΑΡΙΙ Stratagene, La Jolla, USA), from human hippocampus (XZAP, Stratagene, La Jolla, USA), and a cDNA library of human cerebellum (XZAP, Stratagene) are screened by hybridization with the rat mGluR4 fragment. Hybridization is performed in 5x SSC, 0.02% (w / v) Ficoll (Type 400), 0.02% (w / v) polyvinylpyrrolidone, 0.1% (w / v) SDS, 50 mg / mL of Herring Testicle DNA. Prehybridization is performed between 30 mins at 3 hours at 58Â ° C. Hybridization is performed in low stringency at 58 ° C overnight in the same solution containing the 32 P-labeled fragment at a concentration of 1.3x105 cpm / ml. Washings are performed three times for 20 min each at 58øC in 2x SSC / 0.1% SDS.

Phages hybridizing to the rat mGluR4 probe are purified by a second and third round of screening under the conditions described above. The cDNA inserts carried by the purified phage are captured by excision in vivo using the ExAssist / SOLR system (Stratagene, La Jolla, USA). 53 ΡΕ1199359 1.7 Characterization of isolated cDNA clones: some cDNA inserts are characterized by restriction enzyme mapping and DNA sequence analysis. One of these clones, cDNA of cmR20 (isolated from the human cerebellum library) contains an insert of approximately 3.3 kb. Analysis of the cmR20 sequence indicates that it contains the near-complete coding region of human mGluR4 including a translation termination codon (nt 158 to 2739, cf. SEQ ID NO: 1) as well as approximately 750 nt of the 3 'untranslated region. The 5 'end including the absence of the translation start codon. 1.8 Isolation of the 5 'end of human mGluR4: To complete the coding region of human mGluR4, PCR reactions are performed using human genomic DNA or human brain RNA as template for the first strand of cDNA. The sense primer P3 corresponds to the 5 'end of rat mGluR4 cDNA, the antisense primer P4 to nt 440-459 of the rat mGluR4 cDNA.

Table 2 P3: 5'-GCGCTGCAGGCGGCCGCAGGGCCTGCTAGGGCTAGGAGCGGGGG-3 'corresponding to nt 11-37 of rat mGluR4 cDNA [Tanabe et al., Neuron 8: 169-179 (1992)] P4: 5'-GCGGAATTCCCTCCGTGCCGTCCTTCTCG-3' corresponding to nt 440-459 from rat mGluR4 cDNA [Tanabe et al., Neuron 8: 169-179 (1992)]. 54 ΡΕ1199359

Additional sequences are underlined, restriction enzyme sites are indicated in bold.

PCR reactions for a 100 mL reaction mixture are: 400 ng of human genomic DNA, 1 mM of each primer, 2 mM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP and dTTP) in PCR buffer (10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2, 50 mM KCl, and 2 U

AmpliTaq Polymerase. Amplification is performed using the following conditions: 1 min of denaturation at 95øC, 1 min of annealing at 56øC, and 1 min of extension at 72øC, for a total of 32 cycles. Initial denaturation is carried out for 3 min at 94 ° C.

Products from several independent PCRs are digested with the PstI and EcoRI restriction enzymes, purified from the gel, and ligated through thePstl / EcoRI sites of pBluescript SK (Stratagene). Subcloned fragments from several independent PCRs are analyzed by DNA sequence analysis (cR4PCR1-4). Sequence analysis shows that the clone CR4PCR2 encodes 380 nt of hmGluR4 coding region including the translation initiation codon (nt 1-380, cf. SEQ ID NO: 1). The CR4PCR2 overlaps the 3 'end at 223 nt with cmR20. The complete deduced amino acid sequence of the hmGluR4 protein is shown in SEQ ID NO: 2. 55 ΡΕ1199359

Research of human fetal brain and human cerebellum cDNA libraries by low-stringency hybridization using the radiolabelled rat mGluR4 fragment (as described in 1.5 and 1.6) allows the isolation of hMGluR7. cDNA clones that identify the human metabotropic glutamate receptor mGluR7 subtype. Characterization of cDNA clones by DNA sequence analysis reveals that isolated cDNAs represent at least two apparent mRNA mRNA / binding variants of mRNA. The cmR2 cDNA (isolated from a human fetal brain cDNA library) has a size of 3804 nt. Clone cmR2 contains 2604 nt of the hmGluR7 coding sequence including a translation termination codon followed by 1200 nt of the 3 'untranslated sequence (cf. SEQ ID NO: 3). The cmR3 cDNA (isolated from a human hippocampal cDNA library) has a size of 1399 nt (SEQ ID NO: 5). The cmR3 contains 270 nt of the 3 'end coding region of hmGluR7 including a translation termination codon (the deduced amino acid sequence is shown in SEQ ID NO: 6) followed by 1129 nt of the 3' untranslated sequence. The cmR3 sequence is completely contained in cmR2 but differs from cmR2 by deleting the 92 nucleotides that extend from the position of nt 2534 to the position of nt 2625 in SEQ ID NO: 3). This apparent hmGluR7 excision / binding variant produces a 3 'end other than the deduced amino acid sequence of hmGluR7. The cmR5 cDNA (isolated from 56 ΡΕ1199359 a human fetal brain cDNA library) has a size of 1588 nt (SEQ ID NO: 7). The cmR5 cDNA overlaps at 1424 nt with the cmR2 cDNA. Diverge at the 3 'end exactly at the insertion / deletion position of cmR2 / cmR3 92-nucleotide. The additional 164 nt of cmR5 encode intron sequences as indicated by the presence of a conserved excision / binding donor sequence immediately following the cmR5 site and the cmR2 / cmR3 sequence divergence, or represents a third excision / binding variant. The 5 'end coding region of the hmGluR7 DNA lacking in the cDNA clones of cmR2, cmR3, and cmR5 is isolated by a combination of genomic library and PCR techniques. A Lambda-Fix genomic library (Stratagene) is screened with a? CoR1 / SmaI restriction fragment comprising nt 1-1304 of the cmR2 cDNA under high stringency hybridization conditions as described in Sambrook, et al. above. Lambda clones hybridizing to the 5 'end dNA of the cmR2 clone are purified and analyzed by restriction and DNA sequencing analyzes. The complete 5 'end of the coding region of human mGluR7 including 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. It is designated as CR7PCR1 and is presented as SEQ ID NO: 9. The deduced amino acid sequence is set forth in SEQ ID NO: 10. CR7PCR1 overlaps the 3 'end with cmR2 to 392 nt. 57 ΡΕ1199359

The DNA sequences encoding the complete proteins hmGluR7a and b are shown 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 to that of the hmGluR4 subtype of Example 1.

Example 3: cDNA encoding the partial hmGluR6

A single cDNA clone, cmR1, with a 1.0 kb insert is isolated from a human hippocampus library by low stringency hybridization using the hmGluR fragment as described above in example 1.5 and 1.6. Approximately 630 nucleotides are homologous to human mGluR4. Additional sequences at the 5 'and 3' ends of cmR1 apparently encode intronic sequences as indicated by the presence of putative excision / binding site and excision / binding acceptor sequences. The cmRl cDNA identifies a portion of the human metabotropic glutamate receptor subtype hmGluR6 (SEQ ID NOs. 15). The deduced amino acid sequence is shown in SEQ ID NO: 16. The complete coding region of hmGluR6 is isolated by screening for cDNA and genomic libraries under conditions of high stringency with cmR1 cDNA as a probe. Comparison of the deduced 58 ΡΕ1199359 amino acid sequences reveals approximately 70% sequence identity with hmGluR4 of Example 1.

Expression of hmGluR cDNAs in mammalian cells 4.1 Receptor expression plasmids: The cDNAs encoding the full length proteins hmGluR4, hmGluR6, and hmGluR7 above are produced from cDNA fragments and ligated into mammalian expression vectors based on constitutive promoters (CMV, SV40, RSV) or inducible promoters. Examples are pBK-CMV (Stratagene), pBK-RSV (Stratagene), pCMV-T7 (Sibia, Inc.) and pICP4 (Novagen, USA). The full-length cDNA encoding the hmGluR4 subtype is incorporated into the mammalian expression vector pBK-CMV by binding the 5 'end fragment of hmGluR4 (clone cR4PCR2) to cmR20 cDNA at the unique Xholque site located at nt 346- 351 of hmGluR4 cDNA. Specifically, the plasmid pBK-CMV-hmGluR4 is produced by three-way ligation of the cDNA / XhoI fragment of cR4PCR2, the XhoI / NotI fragment of the cmR20 cDNA, and the NotI digested vector pBK-CMV. Plasmid pCMV-T7-hmGluR4 is produced by three-way ligation of the PstI / XhoIde fragment cR4PCR4, the XhoI / EcoRI fragment of cmR20, and the PstI / EcoRI digested pCMV-T7-2 vector. Both expression constructs contain the completed coding region hmGluR4 as well as approximately 750 nt 3 'untranslated desires. 59 ΡΕ1199359

Full-length cDNAs representing two hmGluR7 excision / binding variants, designated hmGluR7a (SEQ ID NO: 12) and hmGluR7b SEQ ID NO: 14), are incorporated into pCMV-T7-2 (SIBIA Inc.) using the clones of overlapping cDNA cmR2, cmR3 and hcR7PCR1. A full-length hmGluR7b expression construct, designated pCMV-T7-hmGluR7b, is prepared by three-way ligation of the PstI / Bsal fragment of hcR7PCR1, the Bsal / Eagl fragment of cmR2 and oPstI / NotI fragment of pCMVT7-2. Plasmid pCMV-T7-hmGluR7b contains the complete coding region of hmGluR7b and 191 nt of 3 'untranslated sequences. To construct a full-length expression construct of hmGluR7a, designated pCMV-T7-hmGluR7a, a 370 pbflindIII / EagI fragment of cmR2 is exchanged with the corresponding cmR3 fragment. The Bsal / EagI fragment of the resulting clone is used for a three-way linkage as described above. Plasmid pBK-CMV-hmGluR6 is produced in an analogous manner using standard techniques (Sambrook et al., Supra). Mammalian cell transfection: Mammalian cells (eg CHO-K1, GH3, American Tissue Type Culture Collection) are adapted to grow in glutamate-free medium (Dulbecco's Modified Eagle's medium without L-glutamate and containing a reduced concentration of 2 mM L-glutamine, supplemented with 0.046 mg / ml proline and 10% dialyzed bovine fetal serum, Gibco-60 ΡΕ1199359 BRL). HmGluR expression plasmids are transiently transfected into cells by precipitation of calcium phosphate (Ausubel, F.M., et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, USA).

Cell lines that stably express hmGluRs are produced by lipofectin-mediated transfection (Gibco-BRL) of CHOK1 cells with hmGluR and pSV2-Neo expression plasmids (Southern and Berg, 1982), a plasmid vector encoding the gene of resistance to G-418. Cells are cultured for 48 hours prior to addition. The medium is replaced every two to three days. Cells surviving the selection of G-418 are isolated and cultured in the selection medium. 32 G-418 resistant clonal cell lines are analyzed six to eight weeks after the initial transfection for hmGluR protein expression by immunoreactivity with the anti-hmGluR7 antibody (immunodetection, cf. 4.3, infra) and functional responses following addition of agonists via radioimmunoassay of cAMP (cf. 5.1, infra).

Likewise, the hmGluR expression constructs pBK-CMV-hmGluR4, pCMV-T7-hmGluR4, pCMV-T7-hmGluR7b and pCMV-T7-hmGluR7a are transiently and stably expressed in mammalian cells (CV1, CHO, HEK293, COS ) according to standard procedures (Ausubel, FM, et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, USA). Transfected cells are analyzed for hmGluR expression by several assays: [3H] -glutamate binding, immunocytochemistry studies using antibodies specific for the hmGluR subtype, and assays detecting a change in intracellular concentration decAMP ([cAMP]) . 4.3 Immunodetection of hmGluR protein expression with antibodies against hmGluR specific for the subtype; Expression of the HmGluR protein is analyzed by immunocytochemistry with antibodies against hmGluR specific for the subtype (see Example 7). 1 to 3 days after transfection, the cells are washed twice with phosphate buffered saline (PBS), fixed with PBS / 4% paraformaldehyde for 10 min and washed with PBS. Cells are permeabilized with PBS / 0.4% Triton X-100, followed by washing with PBS / 10 mM glycine, and PBS. Cells are blocked with PBSTB (1x PBS / 0.1% Triton X-100/1% BSA) for further incubation with antiserum against immunopurified hmGluR (0.5-2.0 mg / mL in PBSTB) for 1 h. After three washes with PBS, the cells are incubated for 1 h with goat anti-rabbit IgG alkaline peroxidase (1: 200 in PBSTB; Jackson Immuno Research). Cells are washed three times with PBS and immunoreactivity is detected with 0.4 mg / ml of naphtholform (Biorad) / 1 mg / ml Fast Red (Biorad) / 10 mM Levamisole (Sigma) / 100 mM Tris / HCl pH 8.8 / 100 mM NaCl / 50 mM MgCl 2 - The staining reaction is stopped after 15 min for subsequent PBS lavage. 2 to 4 cell lines, each homogenously expressing hmGluR4, hmGluR6 or hmGluR7, are identified by immunostaining. 62 ΡΕ1199359

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

Stable cell lines expressing hmGluR4, hmGluR6 and hmGluR7 are used to screen for allosteric agonists, antagonists and modulators. These compounds are identified by binding studies employing [3 H] glutamate and / or measurement of changes in second messenger intracellular levels ([cAMP], [Ca2 +]). 5.1 CAMP Radioimmunoassay: Ligand binding and agonist-induced depression of forscolin-stimulated cAMP accumulation (changes in intracellular cAMP concentration) are analyzed by cAMP radioimmunoassay (Amersham). Cells are seeded in 12-well plates at a density of 0.5-2.0 x 105 cells per well and cultured in 2 to 4 days until a confluent layer of cells is obtained. Cells are washed twice with PBS and incubated for 20 min in PBS containing 1 mM 3-isobutyl-1-methylxanthine (IBMX). Cells are incubated with fresh PBS containing 10 mM forskolin, 1 mM IBMX and a known hmGluR agonist for 20 min. The agonistic effect is discontinued and the cAMP produced by the cells is released by the addition of 1 ml of ethanol-water-HCl mixture (100 ml of ethanol, 50 ml of water, 1 ml of 1 M HCl) after aspirating the drug containing the means. The levels of cAMP are 63 ΡΕ1199359 determined by a cAMP radioimmunoassay involving [3 H] cAMP (Ömersham).

HmGluR subtypes 4, 6 and 7 are negatively coupled to adenylate cyclase when expressed in CHO cells. Binding of agonists leads to an inhibition of forskolin-induced accumulation of cAMP. All subtypes are sensitive to AP-4, meaning that AP 4 has an agonistic effect at a concentration of 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, the cells are plated in single wells, the wells containing a single lamella, or 96-well plates and cultured for 1 to 5 days until a confluent 50-100% layer of cells is obtained. The wells are washed three times with a salt equilibration 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-ΑΜ (or fluro-3-ΑΜ) (Molecular Probes, Inc.) 4.99 ml of BBS, 75 μl of DMSO and 6 , 25 pg Pluronic (Molecular Probes, Inc). Cells are washed 3 times with BBS containing 2 mg / ml of bovine albumin followed by three washes on BBS. After allowing the cells to recover for at least 10 min these are used for microfluorometric measurements of [Ca 2+]. 64 ΡΕ1199359

The cells are transferred to a fluorometric device such as an inverted microscope, a spectrofluorimeter of a fluorescence reader. The fluorescence of the calcium indicator (eg, fura-2 or fluo-3) is induced by light illumination of a wavelength length covered by the excitation spectrum of the dye (fura-2: 340/380 nm, . An increase in intracellular concentration of free calcium ion is monitored 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 EC 50 value in cells, thereby inducing a measurable increase in the intracellular concentration of calcium ion. A test compound is said to be an agonist if it induces a Ca 2+ signal comparable to that induced by glutamate. A test compound is said to be an antagonist if the glutamate induced calcium signal is lower in the presence of the test compound than in the absence of the test compound.

Example 6: HmGluR4, 6 and 7 Chimeric Receptors

Intracellular domains of mGluR1, particularly the second intracellular loop (i2) and the C-terminal region, have been shown to be critical in binding to G protein, which also activates phospholipase C / Ca2 + signaling without altering the pharmacological profile of the receptor (Pin et al. al., 65 ΡΕ1199359

EMBO J. 13, 342-348, (1994)). Conventional PCR mutagenesis techniques are used to exchange the intracellular domains of hmGluRs 4,6, and 7 with corresponding domains of hmGluR1. Stable CHO cell lines are produced with the chimeric expression constructs hmGluR4 / 1, 6/1 and 7/1 allowing the analysis of the influence of modulators of the receptor activity (hmGluRs 4,6,7) using Ca 2+ -dependent assays. The production of a chimeric hmGluR7 / 1 receptor is described below. Chimeric hmGluR4 / 1 and hmGluR6 / 1 expression constructs are produced using analogous cloning and PCR techniques, (i) The expression construct pCMV-hmGluR7b is digested with EagI, thereby releasing the complete insert dDNA. The cDNA is cloned into the iVotl site of pBluescript-Not, a derivative of pBluescript II (Stratagene) in which the polylinker sequences between the unique KpnI and NotI sites are removed. The resulting clone is designated as pBluescript-Not-hmGluR7. (ii) The hmGluR1 transmembrane region is cloned by PCR using primers derived from Masu et al., 1991, supra. The oligonucleotide having the sequence 5'-TATCTTGAGTGGAGTGACATAG-3 '(corresponding to nt 1753 to 1774 of the Masu sequence) is used as a sense primer. The antisense primer has the sequence 5'-ACTGCGGACGTTCCTCTCAGG-3 'corresponding to nt 2524 to 2544 of the Masu sequence. The C-terminal end of the excision / binding variants 1a, 1b and 1c is cleaved by PCR using primers derived from Masu et al., 1991, Tanabe et al., 1992, supra, and Pin et al., 1992 (Proc. Natl Acad Sci, USA, 89, 10331-10335 (1992)), respectively. The 66 ΡΕ1199359 oligonucleotide having the sequence 5'-AAACCTGAGAGGA-ACGTCCGCAG-3 '(corresponding to nt 2521 to nt 2543 of the Masu sequence) is used as a sense primer. The oligonucleotides having the sequence 5'-CTACAGGGTGGAAGA-GCTTTGCTT-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'-TCAATAGACAGT-GTTTTGGCGGTC- 3 'corresponding to nt 2671 to 2694 of the Pin sequence, the antisense primers for hmGluRla, lb and lc, respectively, are used. The PCR fragment is cloned into pBluescript II and sequenced completely. (iii) A chimeric cDNA fragment wherein the i2 loop of hmGluR7a or hmGluR7b (nt 2035 to 2106 of SEQ IDs 11 and 13, respectively) is replaced with the corresponding sequences of hmGluR1 is produced by PCR (as described in Pin et al 1994, supra). The fragment is digested with Sma I and Bgl II which digests at the unique restriction sites flanking the i2 loop. The chimeric SmaI / BglII fragment is exchanged for the Sma1 / BglII fragments of pBluescript-Not-mGluR7. (iv) Further replacement of the C-terminal domain of hmGluR7b or hmGluR7a by the corresponding sequences of the abovementioned hmGluR1 insertion / excision variants is achieved by using the unique restriction sites BglII and SacII that flank the C-terminal end of hmGluR7. (v) The resulting chimeric hmGluR7 / hmGluR1 cDNAs are sequenced and digested with Eagl, thereby releasing the complete cDNAs from pBluescript-Not. For stable expression in CHO cells, the chimeric cDNAs are cloned into the single NtI site of the mammalian expression vector pCMV-T7-2.

Example 7: Production and application of anti-hmGluR antibodies

Peptides corresponding to the deduced C-terminal amino acid sequences of hmGluR4 and hmGluR7 are synthesized and coupled to ovalbumin or Tentagel. Polyclonal antisera are produced in rabbits. Antibodies specific for human mGluR are purified from the antisera by 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 human brain extracts. Antibodies specific for hmGluR4 and hmGluR7, respectively, are used to detect hmGluR receptors on transfected cells and to analyze the cell and subcellular expression profile of the hmGluR receptor proteins in sections of 68 ΡΕ1199359 tissue of human brain material. Antibodies are made against different hmGluR specific peptides consisting of 20 amino acids and fusion proteins expressed in E. coli. Peptides are synthesized by solid phase synthesis coupled to keyhole limpet hemocyanin (KLH) or ovalbumin with glutaraldehyde. PCR fragments containing the entire putative intracellular C-terminal fragment of hmGluRs are cloned as BamHI / EcoRl fragments in the E. coli expression plasmid pGEX-2T (Guan and Dixon, Analytical Biochemistry 192, 262-267 (1991)) producing glutathione-S-transferase (GST) / hmGluR fusion genes. E. coli DH5o cells (Gibco-BRL) carrying the expression plasmids with the GST / hmGluR fusion genes are cultured overnight at 37øC in LB / 100 mg / ml ampicillin medium. Cultures are diluted 1:30 in LB and cultured for 2 h at 30øC. Expression of fusion proteins is induced by treatment with 0.1 mM isopropyl-β-D-thiogalactopyranoside for 3 h at 30 ° C. Cells are harvested by centrifugation at 5000 x g. The fusion protein is isolated using glutathione affinity chromatography.

Deposit Data

Plasmids were deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig on 13 September 1993: ΡΕ1199359 69

Plasmid

Plasmid cmRl; No. cm R 2; access no. DSM 8549 DSM 8550

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: CIBA-GEIGY AG (B) STREET: Klybeckstr. (C) TELEPHONE: +41 61 69 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: (C) 962 991 (ii) TITLE OF INVENTION: human receptor proteins (iii) NUMBER OF SEQUENCES: 16 (iv) COMPUTER READING FORM: (A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS # 1.0, (D) COMPUTER PROGRAM: Patentln Release Version 1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: CELL TYPE: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 1..2739 (D) OTHER INFORMATION: / product = " hmGluR " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 70 ΡΕ1199359 ATG CCT GGG AAG AGA GGC TTG GGC TGG TGG TGG GCC CGG ÇTG CCC CTT 46

MetProGfytysArgGly Leu Gly Trp Trp Trp Aia Arg thy Pro Leu 1 § 10 15 TGC CTG CTC CTC AGC CTT TAC GGC CCC TGG ATQ CCT TCC TCC CTG GGA 96

Cys leu leu Leu Ser leu Tyr Gly Pro Trp Met Pro Ser Ser Leu Gfy 20 25 30 AAG CCC AAA GGC CAC CCT CAC ATO AAT TCC ATC CGC ATA GAT GGG GAC 144

Lys Pro Lys Gly His His Hs Mel Asn Ser fie Arg jfe Asp Gly Asp 35 40 45

ATC ACA CTG GGA GGC CTG TTC CCO GTG CAT GGC CGG GGC TCA GAG GGC 192a Thr Leu Gly Gly Leu Phe Pro Vat His GJy Aig Giy Ser Glu Gly 50 55 60 AAG CCC TGT GGA 6AA CTT AAG AAG GAA AAG GGC ATC CAC CGG CTG GAG 240

Lys Pro Cys Gly Glu Leu Lys Lys Glu Lys Giy Be Hfs Arg Leu Glu 65 70 75 60 GCC ATO CTG TTC GCC CTG GAT CGC ATC AAC MC GAC CCG CAC CTG CTG 288 71 ΡΕ1199359

Ate Met Leu Phe Ala leu Asp Arg as Asn Asn Asp Pro Asp Leu Leu m m 98 CCT AAC ATC ACG CTG GGC GCC CGC ATT CTG GAC ACC TGC TCC AGC GAC 336

Pro Aan te Thr Leu Gty Ala Arg te Leu Asp Thr Cys Ser Arg Asp 1® 105 110

ACC CAT GCC CTC GAG CAG TCG CTG ACC ITT GTG CAG GCG CTC ATC GAG 384

Thr Hts Ate Leu Giu Oln Ser Leu Thr Phe Gin Ate Leu te Glu 110 120 125 AAG GAT GGC ACA GAG GTC CGC TGT GGC AGT GGC GGC CCA CCC ATC ATC 432

LysAsp Gly Thr Glu Vai Arg Cys Gty SerGfy Gfy Pro Pro te te 130 135 140 ACC AAG CCT GAA CGT GTG GTG CGT GTC ATC GGT GCT TCA GGG AGC TCG 480

Thr Lys Pro Glu Arg Will Go Gly Will Be Gty Ser Ser 140 150 1S5 180 GTC TCC ATC ÂTG GTG GCC AAC ATC CTT CGC CTC TTC AAG ATA CCC CAG 528

It will be Met Ala Asn ffe Leu Arg Leu Phe Lys ite Pro Gin 155 170 175 ATC AGC TAC GCC TCC ACA GCG CCA GAC CTG AGT GAC AAC AGC CGC TAC 570

Ite Ser Tyr Ala Ser Thr Ala Pro Asp Leu Ser Asp Asn Sm Arg Tyr 180 185 T90 GAC TTC TTC TCC CGC GTG GTG CCC TCG GAC ACG TAC CAG GCC CAG GCC 824 72 ΡΕ1199359

Asp Pfte Pha Ser Arg Will Be Asp Thr Tyr Gin Aia Gin Ata 135 200 205

ATG GTG GAC ATC GTC CGT GCC CTC MG TCG AAC TAT GTG TCC ACA GTO 872

Evil Go Asp Ret Go Arg Aia Leu Lys Trp Asn Tyr Will Be Thr Go 210 215 220 GCC TCG GAG GGC AGC TAT GGT GAG AGC GOT TGG GAG GCC TTC ATC CAG 720

Aia Ser Giu Giy Ser Tyr Giy Glu Ssr Giy Vai Gtu Aia Pha lie Gin 225 230 235 240

MG TCC CGT GAG GAC GGG GGC GTG TGC ATC GCC CAG TCG GTG AAG ATA 768

Lys Ser Arg Giu Asp Giy Gty Go Cys Jé Ala Gir »Ser Vai Lys Ha 245 250 255

CCA CGG GAG CCC MG GCA GGC GAG TTC GAC MG ATC ATC CGC CGC CTC 816

Pro Arg Glu Pm Lys Aia Giy Giu Phe Asp Lys ile Be Arg Arg Leu 260 265 270 CTG GAG ACT TCG MC GCC AGG GCA GTC ATC ATC TTT GCC AAC GAG GAT 864

Leu Giu Thr Ser Asn Aia Arg Aia Val Phe Ala Asn Giu Asp 275 280 285 GAC ATC AGG CGT GTG CTG GAG GCA GCA CGA AGG GCC MC CAG ACA GGC 912

Asp Ile Arg Arg Leu Giu Aia Arg Arg Asia Gin Thr Giy 290 295 300 73 ΡΕ1199359 CAT TTC TTC TGG AT © GGC TCT GAC AGC TGG GGC ICC AAG ATT QCA CCT 960

His Phe Phe Typical Met Gly Ser Asp Ser Trp Gly Ser Lys Ala Pro 305 310 315 320

GTG CTG CAC CTG GAG GAG GTG GCT GAG GGT GCT GTC ACG ATC CTC CCC 100®

VIA leu Giu Gtu Go Ala Glu Gty Ala Go ThriieleuPro 323 330 335 AAG AGG ATG TCC GTA CGA GGC TTC GAC CGC TAC TTC TCC AGC CGC ACG 1050 lys Arg Met Ser Vai Arg Gty Phe Asp Arg Tyr Phe Ser Arg Arg Thr 340 343 350 CTG GAC AAC AAC CGG CGC AAC ATC TGQ TTT GCC GAG TTC TGG GAG GAC 1104

Leu Asp Asn Asn Arg Arg Asn He Trp Pite AJa GIU Phe Trp Giu Asp 355 360 363 AAC TTC CAC TGC AAG CTG AGC CGC CAC GCC CTC AAG AAG GGC AGC CAC 1152

Asn Phe Hys Cys lys leu Ser Arg Hfc * Ala leu Lys Lys Gly Ser His 370 375 380 GTC AAG AAG TGC ACC AAC CGT GAG CGA ATT GGG CAG GA? TCA GCT TAT 1200

Vat lys Lys Cys Thr Asn Arg Glu Arg Ile Gly Gin Asp Ser Ala Tyr 385 390 395 400 GAG CAG GAG GGG AAG GTG CAG TTT GTG ATC GAT GCC GTG TAC GCC ATG 1248

Glu Gin Giu Gly Lys Go Gin Phe Goes Asp Ala Go Tyr Ala Met 405 410 415 74 ΡΕ1199359 GGC CAC GCG CTG CAC GCC ATS CAC CGT GAC CTG TGT CCC GGC CGC GTG 1298

Gty His Ate his His Met Met Arg Asp Le Cys Pro Gly Afg Go 420 425 430 GGG CTC TGC CCG CGC ATON GAC CCT GTA GAT GGC ACC CAG CTG CTT AAG 1344

Gly Leu Cys Pro Arg Met Asp ProVatAspGyy TiGrGtn Leu Leu Lys 435 440 445 TAC ATC CGA AAC GTC AAC TTC TCA GGC ATC GCA GGG AAC CCT GTG ACC 1392

Tyr ile Arg Asn? Asn Piu? Ser Gly M m Gly Asn Pro Go Thr 450 455 460 TTC AAT GAG AAT GGA GAT GCG CCT GGG CGC TAT GAC ATC TAC CAA TAC 1440

Phe Asn Glu Asn Gly Asp Ala Pro Gly Arg Tyr Asp He Tyr Gin Tyr 455 470 475 480 CAG CTG CGC AAC GAT TCT GCC GAG TAC AAG GTC ATT GGC TCC TGG ACT 1488

Gin Leu Arg Asn Asp Ser Ala Gtu Tyr Lys Vai lie Gly Ser Trp Tfrr 485 490 495 GAC CAC CTG CAC CTT AGA ATA GAG CGG ATG CAC TGG CCG GGG AGC GGG 1538

Asp His Leu His Leu Arg lg Gto Arg Mel His Trp Pro Gly Ser Gly 500 505 510 CAG CAG CTG CCC CGC TCC ATC TGC AGC CTG CCC TGC CAA CCG GGT GAG 1584

Gin Gin Leu Pro Arg Ser Ile Cys Ser Leu Pro Cys Gin Pro Gly Glu 75 ΡΕ1199359 515 520 §25 CGG MG MG ACA GTG MG GGC ATG CCT TGC TGG TGG CAC TGC GAG CCT 1832

Arg lys Lys Thr Va! lys Gly Met Pro Cys Cys Trp Mis Cys Giu Pro 530 535 540

TGC ACA GGG TAC CAG TAC CAG GTG GAC CGC TAC ACC TGT AAG ACG TGT 1880

Cys Thr G! And Tyr Gin Tyr Gin Vag Asp Arg Tyr Thr Cys Lys Thr Cys 545 550 555 560

CCC TAT GAC ATG CGG CCC ACA GAG MC CGC ACG GGC TGC CGG CCC ATC 1728

Pro Tyr Asp Met Arg Pro Thr Giu AsnArgThrGfy Cys Arg Pro #e 565 570 575 CCC ATC ATC AAG CTT GAG TGG GGC TCG CCC TGG GCC GTG CTG CCC CTC 1778

Pro fie fte Lys Leu Glu Trp Gly Ser Pro Trp Ala Va) Leu Pro Leu 580 585 590 TTC CTG GCC GTG GTG GGC ATC GCT GCC ACG TTG TTC GTG GTG ATC ACC 1824

Ph® Leu A Go A Go Gly A Ala Ala Thr Lau Phe Va! Vai lie Thr 535 600 605 TTT GTG CGC TAC MC GAC ACG CCC ATC GTC MG GCC TCG GGC CGT GM 1872

Phe Vai Arg Tyr Ase Asp Th r Prole Vat Lys Ata Ser Gly Arg Gfu 610 615 620 CTG AGC TAC GTG CTG CTG GCA GGC ATC TTC CTG TGC TAT GCC ACC ACC 1920 76 ΡΕ1199359

Leu Ser Tyr Leu Leu Leu Gii le Phe Leu Cys Tyr Ala Thr Thr 625 630 635 640

TTC CTC ATG ATC QCT GAC CCC GAC CTT GGC AGC TGC TCG CTG CGG CGA 1966

Phe leu Uei lie Aia Gfu PmAspleuGly Thr Cya Ser LeuArgArg 645 650 655

ATC TTC CTG GGA CIA GGG ATG AGC ATC AGC TAT GCA GCC CTG ACC 2016 le Phe Leu Giy Leu Gfy Met Ser lie Ser Tyr AJa Aia Leu Leu Thr 660 665 670 AAG ACC AAC CGC ATC TAC CGC ATC TTC GAG CAG GGC MG CGC TCG GTC 2064

Lys Thr Asn Arg lis Tyr Arg lie Phe Glu G n Giy Lys Arg Ser Vai 675 680 685 AGT GCC CCA CGC TTC ATC AGC CCC GCC TCA CAG CTG GCC ATC ACC TTC 2112

Be Pro-Wing Arg Arg Be Ser Pro Wing Be Thu Phe 690 695 700 AGC CTC ATC TCG CTG CAG CTG CTG GGC ATC TGT GTT TGG TTT GTG GTG 2160

Being Leu was Leu Gin Leu Leu Giy was Cys Va! Trp Phe Vai Vai 705 710 715 720 GAC CCC TCC CAC TCG GTG GTG GAC TTC CAG GAC CAG CGG ACA CTC GAC 2208

Asp Pm His His Will Go Asp Phe Gin Asp Gin Arg Thr Leu Asp 725 730 735 77 ΡΕ1199359

CCC CGC TTC GCC AGG GGT GTG CTC AAG TGT GAC ATC TCG GAC CTG TCG 2258

Pro Arg Phe Ala Arg Gly Val Leu Lys Cys Asp le Ser Asp Leu Ser 740 745 750 CTC ATC TGC CTG CTG GGC TAC AGC ATG CTG CTC ATG GTC ACG TGC ACC 2304

Leu He Cys Leu Leu Gty Tyr Ser Met Leu Leu Honey Go Thr Cys Thr 755 760 765 GTG TAT GCC ATC AAG ACA CGC GGC GTG CCC GAG ACC TTC AAT GAG GCC 2352

Go Tyr Ate ite lys Thr Arg Gly Go Pro GJu Tbr Phe Asa Glu Ate 770 775 780

AAG CCC ATT GGC TTC ACC ATG TAC ACC ACT TGC ATC GTC TGG CTG GCC 2400

Lys Pro He GlyPhe ThrMet Tyr Thr Thr Cys lie ValTrpLeuAfa 785 790 795 800

TTC ATC CCC ATC TTC TTT GGC ACC TCG CAG TCG GCC GAC AAG CTG TAC 2448

Pheite Proite PhePhe Gty Thr Ser Gin Ser Ate Asp Lys Leu Tyr 805 810 815 ATC CAG ACG ACG ACG CTG ACG GTC TCG GTG AGT CTG AGC GCC TCG GTG 2498

Hea Thr Thr Thr Thu Leu Thr Vat Be Will Be Leu Be Ate Be Go 820 825 830 ICC CTG GGA ATG CTC TAC ATG CCC AAA GTC TAC ATC ATC CTC TTC CAC 2544

Ser Leu Gly Met Leu Tyr Met Pro Lys Val Tyr Ite Leu Phe Nis 835 840 845 78 ΡΕ1199359 CCG GAG CAG MC GTG CCC MG CGC MG CGC AGC CTC AM GCC GTC GTT 2692

Pro Glu Gtr »Asn Go to Lys Arg tys Arg Ser tu ty $ Ala Go Go 850 355 860 ACG GCG GCC ACC ATG TCC MC MG TTC ACG CAG MG GGC MC TTC CGG 2640

Thr Ala Ala Thr Mat Ser Asn ty Phe Thr Úln Lys 0y Asn Phe Arg 885 870 875 880 CCC MC GGA GAG GCC MG TCT GAG CTC TGC GAG MC CTT GAG GCC CCA 2868

Pro Asn Gly Glu Ala Lys Ser Glu tu Cys Glu Asn tu Glu Ala Pro GCG CTG GCC ACC AM CAG ACT TAC © TC ACT TAC ACC MC CAT OCA ATC 2736

Ala tu Ata Thr Lys Gin ThrTyr Go Thr Tyr Ttir AsnHteAla lie

SOO 905 S1Q

(A) LENGTH: 912 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 79 ΡΕ1199359

Met Pm GJy lys Arg Gly Lao Gly Trp Trp Trp Ala Arg Leu Pro Leu 1 S 10 15

Cys Leu leu Leu Ser Leu Tyr Gly Pro Trp Met Pro Ser Ser Leu Gly 20 25 30

Lys Pm Lys Gly His Pm Hys Met Aso Ser He Arg He Asp Gly Asp 35 40 45

He Thr Leu Gly Gly Leu Phe Pm Go His Gly-Gly Gly Ser Glu Gly 50 55 6

Lys Pro Cys Gly Glu Leu Lys Lys Gtu Lys Glyte His Arg Leu Glu 65 70 75 80

Ala Met Leu Phe Ala Leu Asp Arg Asn Ase Asp Pm Asp Leu Leu 85 00 95

Pio Ase tle Hw Leu Gyy Aia Arg fte Leu Asp Thr Cys Ser Arg Asp 100 105 110

Thr Hfs Ata Leu Glu Glu Ser Leu Thr Phe Go Gin Ala Leu glu 115 120 125

Lys Asp Gly Thr Gtu Vat Arg Cys Gly Ser Gly Gly Pro Pro He fte 130 135 140

Thr Lys Pm Giu Arg Will Go Gly Will He Gly Ata Be Gly Ser Ser 145 150 155 160

He will be He Met Go Wing Asn He Leu Arg Leu Phe lys He Pro Gin 165 170 175 fie Ser Tyr Aria Ser Thr Aia Pro Asp Leu Ser Asp Asn Ser Arg Tyr 80 ΡΕ1199359 180 18S 190

Asp Phe Phe Ser Arg Will Pro Asp Thr Tyr Gin Aia Gin Ala 198 200 205

Mei Vaf Asp ile Vai Arg Ala Leu Lys Τφ Asn Tyr Will Be Thr Go 210 215 220

Ala Ser Glu Gly Ser Tyr Gly Giu Ser Gly Go Glu A Phe ile Gin 225 230 235 240

Lys Ser Arg Giu Asp Gty is Vys Cys ite Ala G! N Ser Vai Lys tie 245 250 255

Pro Arg Giu Pr @ Lys Ala Gly Giu Pi Asp Lys iie i Arg Arg Leu 280 285 270 ·

Leu Giu Thr Ser Aso Ala Arg Ala Vali Ite Phe Ala Asn Giu Asp 275 280 285

Asp ile Arg Arg V i Leu Glu Ala ASa Arg Arg Ala Asn Gin Thr Gly 290 295 300 Hys Phe Phe Τφ Met Gly Ser Asp Ser Trp Gly Ser Lys ite Ala Pro 305 310 315 320

Go Leu His Leu Giu Giu Go Ala Glu Gly Ala Go Thr ile Leu Pr © 325 330 335

Lys Arg Mei Ser Vaf Aig Gly Phe Asp Arg Tyr Phe Ser Arg Arg Thr 340 345 350 thy Asp Asn Asn Arg Arg Asn lie Trp Phe Ata Glu Phe Trp Giu Asp 355 380 365 81 ΡΕ1199359

Mn Phe His Cys Lys Leu Ser Arg Hys A) to Leu Lys Lys Gly Ser Bis 370 37S 380

Go Lys Lys Cys Thr Mn Arg Glu Arg He Gly Gin Asp Ser Ala Tyr 385 390 395 400

Glu Gin Glu Gly Lys Go Gin Phe Goai Asp Aia Go Tyr Ala Met 405 410 415

Gly Hys Ala Leu Hys Afa Mel H Arg Arg Asp Leu Cys Pre Gly Arg Vai 420 425 430

Gly Leu Cys Pro Arg Mel Mp Pro Go Asp Gly Thr Gin Leu Leu Lys 435 440 445

Tyr Ile Arg Mn Go Mn Phe Ser Gly Ite Aia Gly Mn Pro Go Thr 450 455 460

Phe Asn Glu Mn Gly Mp Wing Pro Gly Arg Tyr Mp Ile Tyr Gin Tyr 465 470 475 480

Gin Leu Arg Mn Asp Ser Ala Giu Tyr Lys Val Gly Ser Trp Thr 4S5 490 495

Asp His Leu His tu Arei He Glu Arg Mel H Trp Pro Gly Ser Giy 500 505 510

Gin Gin Leu Pro Arg Ser is Cys Ser Leu Pro Cys Gin Pro Gly Glu 515 520 525

Arg Lys Lys Thr Val Lys Gly Met Pro Cys Cys Trp Hys Cys Glu Pro 530 535 540 82 ΡΕ1199359

Cys Thr Gly Tyr Gin Tyr Gin Go Asp Arg Tyr Thr Cys lys Thr Cys 545 550 555 560

Pro Tyr Asp Met Arg Pm Thr Gly Asn Arg Thr Gly Cys Arg Pro 565 570 575

Pm He is Lys Leu Glu Trp Gly Ser Pro Trp Ala Vai Leu Pro Leu 560 585 590

Phe Leu Wing Vaf Go Gly He Wing Wing Thr Leu Phe Will Go He Thr 595 600 605

Phe VAl Arg Tyr Asn Asp Thr Pm II »Val Lys Ala Ser Gly Arg Gfu 610 615 620

Leu Ser Tyr Leu Leu Leu Gly lie Phe thy Cys Tyr Ala Thr Thr 625 630 635 640

Phe Leu Mel He Wing Glu Pro Asp Leu Gly Thr Cys Ser Leu Arg Arg 645 650 655 Ile Phe Leu Gly Leu Gly Met Ser He Ser Tyr Ale Ala Leu Leu Thr 660 665 670

Lys Thr Asn Arg ile Tyr Arg Ite Phe Glu Gin Gly Lys Arg Ser V 6 660 660 685

Ser Wing Pro Arg Phe He Ser Pro Wing Ser Gin Leu Wing He Thr Phe 690 695 700

Being Leu He Be Leu Gin Leu Leu Gly He Cys Going Trp Phe Vat Go 705 710 715 720

Asp Pm His His Will Go Asp Phe Gin Asp Gin Arg Thr Leu Asp 83 ΡΕ1199359 725 730 735

Pro Arg Phe Aia Arg Gty Go Leu Lys Cys Asp ite Ser Asp Leu Ser 740 745 750

Leu He Cys Leu Leu Gfy Tyr Ser Met Leu Leu Met Vat Thr Cys Thr 755 760 755

Vat Tyr Ala Ite Lys Thr Arg Gty Go Pro Gfu Thr Phe Asn Gtu Ata 770 775 780

Lys ProtYGyPhe Thr Met Tyr Thr Thr Cys ite Vat Trp Leu Ata 785 700 795 800

Phe Ile Pro Phe Phe Gty Thr Ser Gin Ser Ata Asp Lys Leu Tyr 805 810 815 lie Gin Thr Thr Thr Leu Thr Will Be Vat Ser Leu Ser Ata Ser Vat 820 825 830

Ser Leu Gty Met Leu Tyr Met Pm Lys Go Tyr ite lie Leu Phe H 835 840 845

Pm Gfu Gin A $ r »Vat Pro Lys Arg Lys Arg Ser Leu Lys Aia Vat Vat 850 855 860

Thr Aa Ala Thr Met Ser Asn Lys Phe Thr Gin Lys Gly Asn Phe Arg 865 870 875 880

Pro Aso Gíy Giu Aia Lys Ser Gfu Leu Cys Gtu Asn Leu Giu Ata Pm 88S 880 895

AIA Leu Ala Thr Lys Gtrs Thr Tyr Go Thr Tyr Thr Asn Hts Aya ite 800 805 910 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: 84 ΡΕ1199359 (A) LENGTH: 3804 base pairs (B ) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 1..2604 (D) OTHER INFORMATION: / product = "region encoding hmGluR7 of cmR2 " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CCC QTA CAC GCC MG GGT CCC AGC GGA OTO CCC TGC GGC © AC ATC AA 48

Pm Go His Wing Lys Gly FroSerGfy VaiProCysGly Asp Lys 1 8 10 15 AGG GAA AAC GGG ATC CAC AG CTG CM GCC ATC CTC TAC CCC CTG GAC $ 8

Arg GtuAsnGly lie His Arg Leu Glu Afa Met LeuTyrAfa tu Asp 28 25 30 CAC ATC AAC AGT GAT CCC AAC GTA CTG CCC MC GTG ACG CTG GGC GCG 144

Ginie A »! Ser Asp Pm Asn Uu tu Pro Asn Go Tfcr leu Gly Ate 35 40 45 85 ΡΕ1199359 CGG ATG CTG GAC ACT TGT TCC AGG GAC ACT TAC GCG GTC GAA CAG TCG 192

Arg lie tu Asp Thr Cys Ser Arg Asp Thr Tyr Ala Leu Giu Gin Ser 50 55 60 C7T ACT TTC GTC CAG gcg ctc ATC CAG AAG GAC ACC TCC GAC GTG CSC 240

Leu TTir Phe Vsl Gin Ala Leu lie Gin Lys Asp Thr Ser Asp Go Arg 65 TO 75 80 TGC ACC AAC G6C GAA CCG CCG GTT TTC GTC AAG CCG GAG AAA GTA GTT 258

Cys Thr Asn Gly Glu Pm Pm Vat Phe Go Lys Pm GSu Lys Go Go 55 90 95 GGA GTC ATT GGG GCT TCG GGG AGT TCG GTC TCC ATC ATG GTA GCC AAC 306

Giy VafileGty Ala Ser Gíy Ser Ser VaiAleAsn 100 105 110 ATC CTG AGG CTC TTC CAG ATC CCC CAG ATT AGT TAT GCA TCA ACG GCA 384 lie Leu Arg Leu Phe Gin lie Pro Gin l and Ser Tyr Ala Ser Thr Ala 115 120 125 CCC GAG CTA AGT GAT GAC CGG CGC TAT GAC TTC TTC TCT CGC GTG GTG 432

Pro GSu thy Ser Asp Asp Arg Arg Tyr Asp Phe Phe Ser Aig Will Go 130 135 140

CCA CCC GAT TCC TTC CAA GCC CAG GCC ATG GTA GAC ATT GTA AAG GCC m

Pm Pro Asp Ser Phe Gin Ala Gin Wing Go Asp He Go Lys Wing 145 ISO 155 160 86 ΡΕ1199359

CTA GGC TGG MT TAT GTG TCT ACC CTC GCA TCG GM GGA AGT TAT GGA

52S

Leu Giy Τφ Asn Tyr Will Be Tt »r Leu Ala Ser Glu Giy Ser Tyr Giy tas 179 175 GAG AM GGT GTG GAG TCC TTC ACG CAG ATT TCC AAA GAG GCA GGT GGA 576

Glu lys Gly Go Glu Ser Pha Thr Gin lie Ser lys Glu Ala Giy Giy 189 185 199 CTC TGC ATT GCC CAG TCC GTG AGA ATC CCC CAG GM CGC AM GAC AGG 824

Leu Cys and Ala Gin Ser Vaf Arg lie For Gin Glu Arg Lys Asp Arg 195 200 295 ACC ATT GAC TTT GAT AGA ATT ATC AAA CAG CTC CTG GAC ACC CCC MC 672 Tere Re Asp Phe Asp Arg fie He Lys Gin Leu LeuAspThrProAsn 219 215 220 TCC AGG GCC GTC GTG ATT TTT GCC AAC GAT GAG GAT ATA MG CAG ATC 720

Ser Arg AaVatValfte Phe Ala Asn Asp Gtu Asp He Lys Gin 8e 225 230 235 240 CTT GCA GCA GCC AM AGA GCT GAC CM GTT GGC CAT ΤΓΤ CTT TGG GTG 758

Leu Ala Ale Ma Lys Arg Ale Asp Gin Vat Giy Hys Phe Leu Trp Go 245 250 255 GGA TCA GAC AGC TGG GGA TCA AAA ATA MC CCA CT © CAC CAG CAT GM 816

Giy Ser Asp Ser Trp and Ser Lys He Asn Pro leu His Gin HIs Glu 260 285 270 87 ΡΕ1199359 © AT ATC GCA GAA GGG GCC ATC ACC ATT CA © CCC AA © CGA GCC AC © © T © m

Asp ISd AÎ © thi Ma Th Th e ite Gin Pro Lys Arg Ma Thr V a m 280 288 GAA GGG TTT © AT GCC TAC TTT AC © TCC CGT ACA CTT GAA AAC AAC AGA 912 Giu Giy PheAsp AlaTyrPhe Thr Ser Aig Thr tu © fu Asn Asn Arg 290 29S 300 AGA AAT GTA TG © TTT GCC GAA TAC TG © AG GAA AAC TTC AAC TGC AAG 980

Arg Aso Vaf Trp Phe Ala © Tyr Trp © tu © 1u Asn Phe Asn Cys lys 305 310 315 320

TTG ACG ATT AGT GGG TCA AAA AAA GAA GAC ACA GAT CGC AAA TGC ACA 1000 thy Thr Ser Gly Ser Lys Lys G! U Asp Thr Asp Arg Lys Cys Thr 325 330 335 GGA CAG GAG AGA ATT © GA AAA GAT TCC AAC TAT © A © CA © GA © GGT AAA 1058

Gly Gin G Arg Arg He Giy Lys Asp Ser Asn Tyr Giu Gin Glu Gty Lys 340 346 350 © TC CAG TTC GT © ATT GAC GCA GTC TAT © CT AT © GCT CAC GCC CTT CAC 1104

Vei © In Phe Va! Ile Asp Ala Val Tyr EIA Mat act His act viewing His 355 360 365 CCC ATG AAC MG GAT CTC TGT GCT GAC TAC CGG © GT © TC TGC CCA © AG 1152 His Met Asn Lys Asp Leu Cys) Asp Tyr Arg Gly Go Cys Pro Q! U 370 37S 380 ΡΕ1199359 ATG GAG CAA GOT GGA GGC AAG AAG TTG CTG AAG TAT ATA CGC MT GTT 1200

Me »Gin Gin Aty Gys Giyys Lys Leu Uu Lys Tyr ite Arg Asn Go 385 380 395 400 AAT rrc MT GGT AGT GCT GGC AGT CCA GTG ATG TTT MC MG MC GGG 1248

Asm Phe Aso Gty Ser AJa Gty Thr P Va Va Met Met Phe Asn lys Asn G 40 405 410 415

GAT GCA CGT GGG CGT TAT GAC ATC TTT CAG TAC CAG ACC ACA MC ACC 1298

Asp Ata Pro Gty Arg Tyr Aspire Phe Ghi Tyr Gtn ThrTTir Asn Thr 420 425 430 AGC MC CCG GGT TAC CGT GTG ATC GGG CAG TGG ACA GAC GM CTT CAG 1344

Ser Asn ProGy Tyr Arg thy! Gty Gin Trp Thr Asp Giu Leu Gtn 435 440 445

CTC MT ATA GAA GAC ATG CAG TGG GGT AM GGA GTC CGA GAG ATA CCC 1392

Leu Asn ile Giu Asp Met Gtn Trp Gty Lys Gy Go Pro Gtu ile Pro 450 455 450 CCCC TCA GTG TGC ACA CTA CCA TGT AAG CCA GGA CAG AGA MG MG ACA 1440

Ata Ser Vai Cys Thr Leu Pro Cys Lys Pro Gty Gtn Arg lys Lys Thr 465 470 475 450 CAG AM GGA ACT CCT TGC TGT TGG ACC TGT GAG CCT TGC GAT GGT TAC 1488

Gtn Lys Gly Thr Pro Cys Cys Trp Thr Cys Gly Pro Cys Asp Gty Tyr 485 490 455 89 ΡΕ1199359 CAG TAC CAG TTT GAT GAG ATG ACA TGC CAG CAT TGC CCC TAT GAC CAG 1530

Gin Tyr Gfn Phe Asp GJu Met Thr Cys Gin His Cys Pro Tyr Asp Gin 500 505 510 AGG CCC AAT GAA AAT CGA ACC GGA TGC CAG GAT ATT CCC ATC ATC AAA 1554

Arg Pro Asn Glu Asn Arg Thr Gly Cys Gin Asp Ile Protein Ile Lys 515 520 525 CTG GAG TGG CAC TCC CCC TGG GCT GTG ATT CCT GTC TTC CTG GCA ATG 1532

Leu Glu Trp His Ser Pro Trp Ala Pro Pro Go Phe Leu Afa Met 530 535 540 TTG GGG ATC ATT GCC ACC ATC TTT GTC ATG GCC ACT TTC ATC CGC TAC

Leu GSy ile iie Ala Thr lie Phe Met Ala Thr Pha lie Arg Tyr 545 550 555 560 AAT GAC ACG CCC ATT GTC CGG GCA TCT GGG CGG GAA CTC AGC TAT GTT 1728

Asn Asp Thr Pro read will Arg Ala Ser Gly Arg Glu Leu Ser Tyr Go 505 570 575 GTT TTG ACG GGC ATC TTT CTT TGC TAC ATC ATC ACT TTC CTG ATG ATT 1776 Leu Leu Thr Gly Ile Phe Leu Cys Tyr Ile Ile Thr Phe Leu Met ile 580 585 590 GCC AAA CCA GAT GTG GCA GTG TGT TCT TTC CGG CGA GTT TTC TTG GGC 1824

Until Lys Pro Asp Val Until will Cys Ser Phe Arg Arg Vat Phe Leu Gly 595 800 805 90 ΡΕ1199359 TTO GGT ATG TGC ATC AGT TAT GCA GCC CTC TTG ACG AAA ACA MT CGG 1872 thy Gty MetCys Ile SerTyrAia Aia Leu Leu THF Lys Thr AsnArg 619 615 620

ATT TAT CGC ATA TTT GAG CAG GGC MG AM TCA GTA ACA GCT CCC AGA 1920 Ile Tyr Arg Ite Phe Glu Gin Gly Lys Lys Ser ValThrAia Pro Afg 626 $ 39,635,640 CTC ATA AGC CCA ACA TCA CM CTG GCA ATC ACT TCC AGT ΪΤΑ ATA TCA 1888

Leu Ile Ser Pro Thr Ser Gin Leu Ala «and Thr Ser Ser Leu Ile Ser 646 $ 50 $ 65 GTT CAG CTT CIA GGG GTG TTC ATT TGG TTT GGT GTT GAT CCA CCC MC 201 $

Vai Oto Leu Leu Gly Go Pfte ile Trp Phe Gly will Asp Pro Pro Ase 669 665 670 ATC ATC ATA GAC TAC GAT GM CAC MG ACA ATG MC CCT GAG CAA GCC 2064

Ile Ile Ile Asp Tyr Asp Giu Hys Lys Thr Met Asn Pnj Giu Gin Ala 675 680 6 $ 5 AGA GGG GTT CTC AAG TGT GAC ATT ACA GAT CTC CM ATC ATT TGC TCC 2112

Arg Gly Vai Leu Lys Cys Asp ile Thr Asp Leu Gin Ile lie Cys Ser 680 695 700 TTG GGA TAT AGC ATT CTT CTC ATG GTC ACA TGT ACT GTG TAT GCC ATC 2160

Leu Gly Tyr Ser ite Leu tu Met Vai Hir Cys Thr Vai Tyr Wing TOS 710 715 720 91 ΡΕ1199359

AAG ACT CGG GGT GTA CCC GAG AAT TTT AAC GM GCC MG CCC ATT GGA m $ Lys Thr Arg Gly Go pm GTU Am Phe wing GTU Ala Lys Pro ite gfy 725 730 735 TTC ACT ATG TAC ACG ACA TGT ATA GTA TGG CTT GCC TTC ATT CCA ATT 2256

Pha Thr MeI Tyr Thr Thr Cys tt Trp tsu Ata Phe fie Pio fia 740 745 750 TTT TTT GGC ACC GCT CM TC A GCG GM MG CTC TA ACTA CM ACT ACG 2304 The Phe Gly Thr Aia Gin Ser Ata 6 * Ly * thy Tyr i Gin Thr Thr 755 760 765

ACG CTT ACA ATC TCC ATG MC CTA AGT GCA TCA GTOT GCG CTG GGG ATG 2352

Thr thy Thr fie Ser Met As Le Ser Ser Vat Ala le Giy Met 770 775 760 CTA TAC ATG CCG AM GTG TAC ATC ATC ATT TTC CAC CCT GM CTC MT 2400 teu Tyr Met Pio lys Vai Tyr iie Mia Phe His Pro Gfu tu Asm 785 790 795 800 GTC CAG AM CGG MG CCA AGC TTC MG GCG GTA GTC ACA GCA GCC ACC 2448

Go Gin Lys Arg Lys Arg Ser Phe Lys Aia Vaf Go Thr Aia Aia Thr 805 810 815 ATG TCA TCG AGG CTG TCA CAC AM CCC AGT GAC A6A CCC MC GGT GAG 2498

Met Ser Ser Arg Arg Ser His tys Pro Ser Asp Arg Pro Asn Gly Giu 820 825 880 GCA MG ACC GAG CTC TGT GM MC GTA GAC MC MC MC TGT ATA CCA 2544 92 ΡΕ1199359

Ata lyt Thf Gtu Leu Cys Oto Aen Go Asp PmAsrtAsnCys Se Pto 335 840 848 CCA GTA AGA AAG AGT GTA CAA AAG TCT GTT ACT TOO TAC ACT ATC CCA 2502

Pm Go Arg Lys Be Go Gto Lys Be Go Thr Trp Tyr Thr ite Pm 850 855 880 CCA ACA GTA TAGCTTTTGA CTOCTnCCC AAAGGCCCT6 CTGCAAAAM 2041

Pm Thr Val 865 GAAGTATGTC AGTTATAATA ACCTGGTTAT CTAACCT6TT CCATTCCATG GMCCATGGA 2701 GGAGGAAGAC CCTCAGTTAT TTTGTCACCC AACCTGGCAT AGGACTCTTr GGTCCTACCC 2761 GCTTCCCATC ACCGGAGGAG CTTCCCCGGC CGGGAGACCA GTGTTAGAGG ATCCAAGCGA 2821 CCTAAACAGC TGGTTTATGA AATATCCTTA CTTTATCTGG GCTTAATAAG TCACTGACAT 2881 CAGCACTGCC AACTTGGCTG CAATTÔTGOA ccttccctac CAAAGGGAGT GTTGAAACTC 2041 AAGTCCCGCC CCOGCTCTIT AGAATGGACC ACTGAGAGCC ACAGGACCGT TTTGGGGCTG 3CKS1 ACCTGTCTTA TTACGTATGT ACTTCTA60T TGCAAGorrr rGAAATTTTC TGTACAGTTT 3061 93 ΡΕ1199359

GTGAGGACCT TGTTTTCGAA TTGCACTTTG 3121 CCATCTGATÔ TCGTACCTCG GTTCACTGTT TGCCTTGTTT AAATTTTAAA TCATAGAGCC 3181 CTATTCTCTC AGACGGTGGA ATATTT6GAA ACAA7TAAAA ACATGACTGT TTTTAAAGCA 3241 ATCTTGGCAG ACTAAAACAA 6TACATCTGT ATAATTACGT AAAAGATGTT TATAGTACCA 3301 CTGCACATCA TGTTTTTTTT TTTAAGACAA

TAAAGACCAA ATGATAGGTT AAACTGTGCT 3381 GAGNAAGTAT GCCCCACCTA TCTTTNGNAT ACATAAAAGG ATGCATGCCA AAGGTATTGG 3421 CTGAACTGNA TAGAGGTCTT GATCTTTGGA GTAATGTATT TCTCTTTTGT TACAGTACAT 3481 GTTTATTATG TTCAATATTT GTATTTGTGT ΤΑΤΠΤΤΑΑΤ TAGNGTATAT GAATATTTTG CAA7AATTTT AATAATTATT AAGCTGTTTG 3641 AAGGAAAGAA TTGACTGATC TATGGATTTT 3881 TCATGTCTTG AGGTTTTGrr CATGCCCCCT AGTGTGATAA TAATAAGTAC GGACTTTAGG 3661 AAAAAAAGCA TGTATGTTTT TTAGTGTTTG TTTCGTTAAT TTGCTGAAAA CTTGCTGCTT 3721 ATGTGCCAAT TTAGTSGAM AGAACAACCC ATTCCCTCTT TCCATTCTCT TTCAATTCTG TGATATTGTC CMGMTGTA TCAATAAAAT 3781 3804

ACTTGGTTA ACTTTAAAM AM (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 867 amino acids 94 ΡΕ1199359 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Pro Go HSs A! To Lys Giy Pro Be Giy Go Pm Cys Giy Asp lie Lys 15 10 15

Arg Giu Asn Giy lie His Arg thy Giu Ata Met thy Tyr Aia tu Asp 20 25 30

Gin lie Asn Ser Asp Pro Asn Leu Leu Pro Asn Go Thr Leu Giy Aia 35 40 45

Arg Ile Leu Asp Thr Cys Ser Arg Asp Thr Tyr Aia Leu Giu Gin Ser 50 55 60

Leu Thr Phe Vai Gin Aia Leu iie Gin Lys Asp Thr Ser Asp Vai Arg S5 70 75 80

Cys Thr Asn Giy Gíu Pro Pm Go Phe Go Lys Pm Giu Lys Go Go 85 30 35 95 ΡΕ1199359

Giy Vai Ife Giy Aia Ser G! Y Ser Ser Vai Ser lie Met Vai Aia Asn 100 105 110

He Leu Arg tu Phe Gin He Pro Gin He Ser Tyr Ate Ser Thr Aia 115 120 125

Pro Gtu Leu Ser Asp Asp Arg Arg Tyr Asp Phe Phe Ser Arg Go VáJ 130 135 140

Pro Pro Asp Ser Phe Gin Aia Gin Honey Wing Go Asp t! E Vai Lys Aia 145 150 155 160

Leu Giy Trp Asn Tyr Will Be Thy Thy Aia Be Giu Giy Ser Tyr Giy 165 170 175

Gtu Lys Giy Go Glu Ser Phe Thr Gin ife Ser Lys Glu Aia Giy Giy 180 185 190 thy Cys ife Aia Gin Ser Vai Arg He Pm Gin Glu Arg Lys Asp Arg 195 200 205

Thr He Asp Phe Asp Arg He He Lys Gin Tu Leu Asp Thr Pro Asn 210 215 220

Ser Arg Ata Vai He Phe Ala Asn Asp Glu Asp He Lys Gin iie 225 230 235 240

Leu Wing Wing Aia Lys Arg Wing Asp Gin Wing Giy His Phe your Trp Go 245 250 255

Giy Ser Asp Ser Τφ Giy Ser Lys iie Asn Pro seu His Gin His Gíu 260 265 270

Asp He Aia Glu Giy Ala iie Thr tle Gin Pro Lys Arg Aia Thr Go 96 ΡΕ1199359 275 280 285 Gly Gly Phe Asp Ala Tyr Phe Thr Ser Arg Thr Leu Giu Asn Asn Arg 290 295 300

Arg Asn Go Trp Fhe Ala Giu Tyr Trp Glu G Asu Phe Asn Cys Lys 305 310 315 320 thy Thr Ite Ser Gly Ser Lys Lys Giu Asp Thr Asp Arg Lys Cys Thr 325 330 335

Gly Gin Giu Arg Ha GSy Lys Asp Ser Asn Tyr Giu Gin Giu Gly Lys 340 345 350

Go to Gin Phe Vai ile Asp Ala Vai Tyr Aa Met Ala Hfs Ala Leu His 355 380 385

His Met Asn Lys Asp Leu Cys AJa Asp Tyr Arg Gfy Goes Cys Pro Giu 370 375 380

Met Giu Gin Wing Gly Gly Lys Lys Leu Leu Lys Tyr Ile Arg Asn Go 385 390 395 400

Asn Phe Asn Gly Ser Ala Gly Thr Pro Go Mel Phe Asn Lys Asn Gly 405 410 415

Asp Aia Pro Gly Arg Tyr Asp lie Phe Gin Tyr Gfn Thr Thr Asn Thr 420 425 430

Ser Asn Pm Gly Tyr Arg Leu ile Gly Gin Trp Thr Asp Giu Leu Gin 435 440 445

Leu Asn Ile Giu Asp Met Gin Trp Gly Lys Gly Vai Arg GIu Ile Pro 450 455 460 97 ΡΕ1199359 Aía Ser Va! Cys Thr Leu Pm Cys lys Pm Gly Gin Arg Lys lys Thr 465 470 475 460 Gin Lys Gly Thr Pm Cys Cys Trp Thr Cys Gtu Pm Cys Asp Gty Tyr 485 400 495

Gin Tyr Gin Phe Asp Glu Met Thr Cys Gin His Cys Pro Tyr Asp Gin 500 505 510

Arg Pm Asn Glu Asn Arg Thr Gly Cys Gin Asp Ile Proline Ile 515 520 525

Leu Gtu Trp His Ser Pro Trp Ate Vai ile Pro Vai Ph® leu Ala Met 530 535 540

Leu Gly 11® J Ata Ata Thr Ile Phe Vai Mei Ala Thr Phe Ile Arg Tyr 545 550 555 560

Asn Asp Thr Pr® lie Val Arg Arg Ser G Arg Arg Leu Ser Tyr Val 565 570 575

Leu Leu Thr Gly Ile Phe Leu Cys Tyr II® Ile Thr Phe Leu Met i 580 585 590

Aia Lys Pr® Asp Go Ate Go Cys Ser Phe Arg Arg Go Phe Leu Giy 595, 600 605

Leu Gly Met Cys ite Ser Tyr Aa Ala Leu Leu Thr Lys Thr Asn Arg 610 615 620 ile Tyr Arg lie Phe Glu Gin Gly lys Lys Ser Vai Thr Ate Pro Arg 625 630 635 840 98 ΡΕ1199359

Leu Irie Ser Pr © Thr Ser Gin Leu Ala He Thr Ser Ser Leu He Ser 645 650 655

Go Gin Leu Leu Giy Go Pise He Trp Phe Gfy Go Asp Pro Pro Asn 660 665 670

Ite I He He Asp Tyr Asp Glu Hys Lys Thr Met Asn Pro Gtu Gin Ala 675 680 666

Arg Gfy Go Leu Lys Cys Asp He Thr Asp Leu Gin ile He Gys Ser 6S0 665 700

Leu Giy Tyr Ser Ite Leu Leu Met Go Thr Cys Thr Go Tyr Ala Ite 705 710 715 720

Lys Thr Arg Gfy Pro Glu Mn Phe Asn Gtu Ala Lys Pm Ite Giy 725 730 735

Phe Thr Met Tyr Thr Thr Cys Ite Go Trp Leu Ala Phe Ite Pte 740 745 750

Phe Phe Giy Thr Ala Gin Ser Ala Glu Lys Leu Tyr Ite Gin Thr Thr 755 760 765

Thr Leu Thr Ile Ser Met Asn Leu Ser Ate Le Val Lea Giy Met 770 775 780

Leu Tyr Met Pro Lys Go Tyr Ite ite ite Phe Pro Glu Leu Asn 785 790 795 800

Go Gin Lys Arg Lys Arg Ser Phe Lys Ala Go Go Thr Ate Ala Thr 805 810 815

Met Ser Ser Arg Arg Leu Ser Hos Lys Pro Ser Asp Arg Pro Asn Gly Glu 99 ΡΕ1199359 820 825 830

Alysis Thr Giu le Cys Glu Asn Go Asp Pro Asn Aso Cys lie Pro 835 840 845

Pro Vai ArglysSer Go Glr »lys Ser Vai Thr Trp Tyr Thr He Pro 860 855 860

ProThrVaf 885 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1399 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 1..270 (D) OTHER INFORMATION: / product = "region encoding hmGluR7 of cmR3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ATC ICC ATG AAC CTA AGT OCA TCA GTG 6CG CTG GGG ATG CTA TAC ATG 48μL Ser Met Asn Your Ser A Ser Ser A Leu Gly Met leu Tyr Mst 1 5 10 15 CCG AAA GTG TAC ATC ATC ATT TTC CAC CCT GAA CTC AAT GTC CAG AAA 98 100 ΡΕ1199359

Piro Lys Vai Tyr lie i lie Phe His Ρκ »Ohi Uu Asn Go Gin Lys 20 25 30 CGS AAG CGA AGC TTC AAG GCG GTA GTC ACA GCA GCC ACC ATG TCA TCG 144

Aig Lys Αφ Ser Phe lys Ala Will Go 17 »Afa Ma Tftf Honey Serar 35 40 45

AGG CT © TCA CAC AM CCC AGT GAC AGA CCC AAG GGT GAG GCA AAG ACC 192

Arg thy Ser His Lys Pro Ser Asp Arg ProAsníayGluAJaLysThr 50 55 60 GAG CTC TGT GAA AAC GTA GAC CCA AAC AGC CCT GGT GCA AAA AAG AAG 240

Gtu tu Cys GluAsnVaí Asp Pm Asn Ser PK »Ala Ata Lys Lys Lys 65 70 75 80 TAT GTC AGT TAT MT AAC CTG GTT ATC TMCCTGTTC CATTCCATGG 287

It will Tyr Tyr Asn Ser Asn Leu Val Ile O5 90 347 AACCATGGAG GGACTCTTTG GAGGAAGACC CTCAGTTATT TTGTCACCCA ACCTGGCATA GTCCTACCCG TGTTAGAGGA crrcccATCA 407 CCGGAGGAGC TTCCCCGGCC GGGAGACCAG TCCMGCGAC CTTAATAAGT CTAMCAGCT 457 GCTTTATGM ATATCCTTAC TTTATCTGGG CACTGAÇATC AAAGGGAGTG AGCACTGCCA 527 101 ACTTGGCTGC MTTGTGGAC cttccctacc ΡΕ1199359 TTGAAACTCA AOTCCCQCCC CGGCTCTTTA SAATGGACCA CTGAGAGCCA 587 CAGGACCGT7

TT < 3 < 30 < 3CTGA CCTGTCTTAT TACGTATGTA CTTCTAOQTT GCAAGGTTTT

GAAATTTTCT 547 GTACA6VTTG TTCACTGTTT TGAGGACCTT 707 TGCACTTTGC CATCTGATGT CGTACCTCGG GTTTTCGAAT TATTTGOAAA GCCTTGTTTT 767 CATAGAGCCC TATTCTCTCA GACGGTGGAA AATTTTAAAA TACATCTGTA CAATTAAAAT 327 TTTAAAGCAA TCTTGGCAGA CTAAAACAAG CATGACTOTA TTAAGACAAA - TAATTACGTT 887 ATAGTACCAC TGCACATCAT GTTTTTTTTT AAAGATGTTT CTTTNGfiATA AAAGACCAAA 947 AACTGTÔCTG AGMAAGTATG CCCCACCTAT TGATAGSTTA ATCTTTGGAA CATAAAAGGA 1007 AGGTATTGGC TGAACTGMAT AGAGGTCTTG TGCATGCCAG TA7TTÔTGTT TAATGTATrr 1087 ACAGTACATG TTTATTATGT TCAATATTTG CTCTTTTGTT ATTTTTAATT AGNGTATATG AATATTTTGC AATAATTTTA ATAATTATTA 1127 AGCTOTTTGA ATGCCCGCTT AGGAAAGAAT 1187 ATGGATmr CATGTCTTGA GGTTTTGTTC TÕACTGA7CA TACTGTTTGT GTGTGATAAG 1247 GACTTTAGGA AAAAAAGCAT GTÃTGTTTTT aataagtact GAACAACCCT TTCGTTAATC 1307 TTGCTGCTTA TGTGCCAATT TAGTGGAAAA TGCTGAAAAÃ rrcccTCTrr CCATTCTCTT TCAATTCTGT GATATTGTCC AAGAATGTAT 1367 CAATAAAATA CTÍTGGTTAA CTTTAAAAAA AA 1399 (2) INFORMATION FOR SEQ ID NO : 6: 102 ΡΕ1199359 (i) CHARACTERISTICS OF SEQUENCE: (A) LENGTH: 89 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Vat Ala Leu Gty Mel Leu Tyr Met 1 S 10 15

Pro Lys VaI Tyr tle ite ife Phe His Pro Glu Leu Asn Go Gin Lys 20 25 30

Arg Lys Arg Ser Phe Lys Ala Vai Hw Ala Aia Tftr Met Ser Ser 35 40 45

Arg Leu Ser His Lys Pro Ser Asp Arg Pro Asn Gly Glu Ala Lys Thr SO 55 60

Glu Leu Cys Glu Asn Go Asp Pro Asn Ser Pro Aia Ala Lys Lys Lys 65 70 75 SO

(1) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1588 base pairs (B) TYPE: nucleic acid (C) STRAND TYPE : single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 2..1447 codes (D) OTHER INFORMATION: / product = " hmGluR7 which portion of cmR5 " SEQ ID NO: 7: SEQ ID NO: 7: SEQ ID NO: 7: SEQ ID NO: 7: SEQ ID NO: 7: SEQ ID NO: 7: G MC AAG GAT CTC TGT GCT GAC TAC CG3 GGT TC TCG CCA GAG ATG 46 Asn lys Asp thy Cys Ale Asp Tyr Arg Gly V Cys Pro Glu Mel 15 10. 15 GAG CAA GCT GGA GGC MG AAG TTG CTG AAG TAT ATA CGC MT GTT AAT 34

GTIGInAlyGlyGly lyslysUuUtilysTyrlleArgAsn ValAsr »20 23 30 TTC MT GGT AGT GCT GGC ACT CCA GTG ATS TTT MC MG MC GGG GAT 142

Phs Asn Giy Ser Ala Gly Thr Pro mt Phe Asn lys Asn Gly Asp 35 40 4 $ GCA CCT GGG CGT TAT GAC ATC TTT CAG TAC CAG ACC ACA MC ACC AGC 100

Ala Fw * Gly Arg Tyr Asp lie Phe Gin Tyr Gl «Thr Thr Asn Thr Ser 30 55 60 MC CC6 GGT TAC CGT CTG ATC GGG CAG TGG ACA GAC GM CTT CAG CTC 236

Asn Pm Gly Tyr Arg leu lt Gly Gin Trp Thr Asp Gp Leu G th ng 65 70 75 AAT ATA GAA GAC ATG CAG TGG GGT AM GGA GTC CGA 6AG ATA CCC GCC 288

Asn I IP Glu Asp Mel Gin Trp Gly Lys Gly VaI Arg Glu Ile Pro Ala 80 85 00 95 TCA GTG TGC ACA CTA TGT AAG CCA GGA CAG AGA AAG MG ACA CAG 334

Ser is Cys Thr thy Pro Cys Lys Pro Gly Gin Arg lys lys Thr Gin 11 105 105 AM GGA ACT CCT TGC TGT TGG ACC TGT GAG CCT TGC GAT GGT TAC CAG 382 lys GlyThr Pro Cys Cys Trp Thr Cys Glu Pro Cys Asp Gly Tyr Gin 118 120 128 TAC CAG TTT GAT GAG ATG ACA TGC CAT CAT TGC CCC TAT GAC CAG AGG 430

Tyr GM Phe Asp Or Mel Thr Cys Gin His Cys Pro Tyr Asp Gin Arg 130 135 140 CCC MT GAA MT CGA ACC GGA TGC CAG GAT ATT CCC ATC ATC AM CTG 478 104 ΡΕ1199359

Pro Asn 6fci Asn Arg Thr Giy Cys Glu Agp 8e Pm Se lys Leu 145 1SO 18 $ GAG TEG CAC TCC CCC TGG GCT GTG ATT CCT GTC TTC CTG GCA ATG TTG 526

Glu Trp Hts Ser Pro Trp Aya Go Pro Phe leu Ate Met thy 160 166 170 178 6GG ATC ATT GCC ACC ATC TTT GTC ATS 3CCACT TTC ATC CGC TAC AAT $ 74

Giy lie lie Ala Thr «and Ptw Vai Met Ala Thr Phe lie Arg Tyr Asn 180 16 $ 190 GAC ACG CCC ATT GTC CGG GCA TCT GGG CGG GAA CTC AGC TAT ôTT CTT 622

Asp Thr Pro Be V al Atg Ala SerGtyArg Glu le Ser Ser Tyr Val leu 195 200 20 $ TTG ACG GGC ATC TTT CTT TCC TAC ATC ATC ACT TTC CTG ATG ATT GCC 670

Leu Thr Giy lie Phs Leu Cys Tyr ile lla Thr Phe leu Met lia 210 215 220

AAA CCA GAT GTG GCA GTG TGT TCT TTC CGG CGA GTT TTC TTG GGC TTG 718

Lys Pro Asp Go Ala Go Cys Be Pbs Arg Arg Go Phe Leu Giy Leu 225 230 23 $ GGT ATG TGC ATC AGT TAT GCA GCC CTC TTG ACG AAA ACA AAT CGG ATT 768 Giy Met Cys tte Ser Tyr Arfa Aia Leu Leu Thr lys Thr Aso Arg lie 240 245 260 255 TAT CGC ATA TTT GAO CAG GGC AAG AAA TCA GTA ACA GCT CCC AGA CTC 814

Tyr Ag Ha Pfte Glu Gto Gty Lys Lys Ser Vai Thr Aia Pro Arg leu 260 26S 270 105 ΡΕ1199359 ATA AOC CCA ACCA CAA CTG GCA ATC ACT TCCAGTTTA ATA TCA GtT 862 Ser Pio Thr Ser Gin Lati Ata te Thr Ser Ser leu fie Ser Vat 275 280 285 CAG CTT CTA GGG GTG TTT ATT TGG TTT GGT GTT GAT CCA CCC AAC ATC 910

Gin Leu read Giy Vai Phe lie Tip Phe Gíy Vaf Asp Pro Pro Asn tle 230 295 300 ATC ATA GAC TAC GAT GAA CAC AAG ACA ATG AAC CCT GAG CAA GCC AGA 958

Asp Tyr Asp G! u His Lys Thr Met Asn Pro Gfu Gin Aria Arg 305 310 31S GGG GTTCTCAAGTGT GAC ATT ACA GAT CTC CAA ATC ATT TQC TCC TTG 1006 Gty Go Lsu Lys Cys Asp t Thr Asp Leu Gin tb Cys Ser Leu 320 325 330 335 GGA TAT AGC ATT CTT CTC ATG GTC ACA TGT ACT GTS TAT GCC ATC AAG 1054

GiyTyrSerte Leu Leu Met Go Thr Cys Thr Vaf Tyr Ala te Lys 340 345 350 ACT CGG GGT GTA CCC GAG AAT TTT AAC GAA GCC AAG CCC ATT GGA TTC 1102

Thr ArgGJy Vaf P? O Gft? AsnPheAso Giu Ata Lys Pio te Gfy Phe 355 300 365 ACT ATG TACACG ACA TGT ATA GTA TGG CTT GCC TTC ATT CCA ATT TTT 1150

Thr Met Tyr Thr Thr Cys te Trp Leu Ala Phe tePro te Pte 370 375 380 TTT GGC ACC GCT CAA TCA GCG GAA AAG CTC TAC ATA CAA ACT ACC ACG 1198

PteGfyThrAfeGfftSerAtaGfuLysteuTyrteGtaThrThrThf 106 ΡΕ1199359 386 380 395 CTT ACA ATC TCC ATO AAC CTA AGT GCA TCA GTG GCG CTG GG6 ATG GTA 1248

Leu Thrite Ser Ser Asn Leu Ser Ala Ser Lea Leu Gly Met Leu 400 405 410 415 TAC ATG CCG AAA GTG TAC ATC ATC ATT TTC CAC CCT GAA CTC ΑΑΤ GTC 1294 Tyr Met Pro Lys Vst Tyr ile He Phe m Pm Giu Leu Asn Go 420 425 4®) CAG AAA CG6 AAG CGA AGC TTC AAG GCG GTA GTC ACA GCA GCC ACC ATG 1342

Gin Lys AfO Lys Arg Ser Pfte Lys AI »Will Go Thr Ata A * to Thr Met 438 440 445 TCA TCS AGG CTG TCA CAC AAA CCC AGT GAC AGA CCC AAC GGT GAG GCA 1390

SerSerArg Uu Ser His Lys Pro Ser Asp Ar $ Pro Asn ô! Y GíAia 450 455 480 AAG ACC GAG CTC TGT GAA AAC GTA GAC CCA AAC AGT GAG AAG TGC AAC 141 »

Lys Thr Glu Leu Cys Glu Asrt Go Asp Pro Asn Ser Gtu Lys Cys Asn 465 470 475

TGC TAC TGACCATCTG CACTGGCATC TAGTCAAGCG ATTGTCTGAG GAAAGGATTT 1494

Cys Tyr 480 TGGAGATTCC CATCTGATAT TCTTCTATTT GGTCTCTTOT ACCCATTTCTCCCATCCTGTACC 1854 ACACATTAATA AAGTTTAAGA ATGTCMGCA AAAG 1588 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 481 amino acids (B) TYPE: amino acid (D) TOPOLOGY : linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 107 ΡΕ1199359

Asn Lys Asp Leu Cys Ala Asp Tyr Arg GSy Go Cys Pro Giu Met Gfu 15 10 15

Gin Wing Gly GSy Lys Lys Leu Leu Lys Tyr He Arg Asn Go Asn Phe 20 25 30

Asn Gly Ser Ala Gly Thr Pro Go Met Phe Asn Lys Asn Gly Asp Ala 35 40 45

Pm Gly Arg Tyr Asp He Phe Gin Tyr Gin Thr Thr Asn Thr Ser Asn 50 55 80

Pm Gly Tyr Arg Leu ife Gly Gin Trp Thr Asp Giu Leu Gin Leu Asn 65? 0 75 80

He Qtu Asp Mel Gin Trp Gly Lys Gly V A Glu Ile Pro A Ser 85 90 95

Go Cys Thr Leu Pm Cys Lys Pro Gly Gin Arg Lys Lys Thr Gfn Lys 100 105 110 108 ΡΕ1199359 Gly Thr Pro Cys Cys Trp Thr Cys Giu Pro Cy $ Asp Gly Tyr Gin Tyr 115 120 125

Gin Phe Asp Giu Met Thr Cys Gin Hls Cys Pro Tyr Asp Gin Arg Pro 130 135 140

Asn Giu Asn Arg Thr Giy Cys Gin Asp lla Pro ile Ita tys Leu Giu 145 150 155 160

Trp His Ser Pro Trp Aaa 11 Pm Go Phe your Ala Met Leu Gly 165 170 175 ff Alfa Thr II Phe Vai Met Aia Thr Phe I Arg Tyr Asn Asp 180 185 100

Thr Prole Go Arg Arg Ser Gly Arg Glu thy Ser Tyr Go your tu 105 200 205

Thr Gly f Phe t Cys Tyr I lie Thr Phe Leu Met I A t tys 210 215 220

Pro Asp Go Aia Go Cys Be Phe Arg Arg Go Phe your Gly thy Gly 225 230 235 240

Met Cys He Ser Tyr Aa Ala tu Leu Thr tys Thr Asn Arg ty Tyr 245 250 255

Arg lie phe Giu Gin Gly tys tys Ser Vai Thr Aia Pro Arg tu lí 260 260 270

Ser Pro Thr Ser Gin Leu Aia Thr Ser Ser Leu lie Ser Vai Gin 275 280 285 thy thy Gly Goes Phe «and Trp Phe Gly Go Asp Pro Pro Asn iie fie 109 ΡΕ1199359 290 295 300 lie Asp Tyr Asp Giu Bis Lys Thr Met Mn Pm Giu GJn Ala Mg ôly 305 310 315 320

Go Leu Lys Cys Asp 1 Thr Asp Leu Gin 3 and Ile Cys Ser Leu Giy 325 330 335

Tyr Ser is Leu Leu Met Val Thr Cys Thr Val Tyr Ala lie Lys Thr 340 345 350

Arg Gly and Pro Glu Mn Phe Asn Gly Ate Lys Pma Gly Phe Thr 355 350 365

Met Tyr Thr Thr Cys He Trp Leu Ate Phe lie Pro ite Phe Phe 370 375 350

Giy Thr Aia Gin Be Ala Giu Lys Leu Tyr He Gin Thr Thr Thr Leu 385 390 395 400

Thr He Ser Met Mn Leu Ser Aia Ser Goa Ala Leu Gly Met Leu Tyr 405 410 415

Met Pro Lys Vai Tyr lie He lie Phe His Pro Giu Leu Asn Vai Gin 420 425 430

Lys Arg Lys Arg Ser Phe Lys Ays Will Go Thr Ala Ate Thr Met Ser 435 440 445

Ser Mg Leu Ser His Lys Pro Ser Asp Arg Pro Asn Gly Glu Ala Lys 450 4S5 460

Thr Glu Leu Cys Glu Mn Go Asp Pro Mn Ser Glu Lys Cys Mn Cys 465 470 475 460

Tyr 110 ΡΕ1199359 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 558 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: linear (ii) MOLECULE TYPE: cDNA (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 1..558 (D) OTHER INFORMATION: / product = " hmGluR7 encoding portion of CR7PCR1 " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

ATG GTC CAG CTG AGG MG CTG CTC CGC GTC CTG ACT TTG ATS MG TTC 48

Met Vai Gla Leu Arg Ly® Leu Leu Arg Vai Leu Thr Leu Met Lys Phe 1 8 10 15

CCC T6C TGC GTG CTG SAG GTG CTC CTG TGC GCG CTG GCG GCG GCG GCG 98 f> Cys Cys Vsi Leu Gtu Go Leu Leu Cys Me Leu Me Ma Ale Me 20 25 30 CGC GGC CAG GAG ATG TAC GCC CCG CAC TC A ATC CGG ATC GAG GGG GAC 144

Am Gty Gin Giu LfetTyrAiaPrcHisSertteArgiíeGiuGíyAsp 111 ΡΕ1199359 35 48 45

GTC ACC CTC GGG GGG CTG TTC CCC GTA CAC GCC MG GGT CCC AGC GGA 132

Go Thr read Gty Giy tu Phe Pro Go Hls Ala Lys Gty Pro Ser Giy 50 5S 60 GTG CCC TGC GGC GAC ATO AAG AGG GM AAC GGG ATC CAC AGG CTG GAA 240 Pn> Cys Giy Asp fie Lys Arg GJu Asn Giy Ra His Arg tu Gltt 65 70 75 80

GCG ATG CTC TAC GCC CTG GAC CAG ATC AAC AGT GAT CCC AAC CTA CTG 288 AIA Met leu Tyr Ala leu Asp Gin I te Asn Ser Asp Pto Asn leu leu 88 90 35 CCC AAC GTG ACG CTG GGC GCG CGG ATC CTG GAC ACT TGT TCC AGG GAC 336

Pro Asn Go Thr read Gty Aia Arg He thy Asp Thr Cys Ser Arg Asp 100 105 110 ACT TAC GCG CTC GM CAG TCG CTT ACT TTC GTC CAG GCG CTC ATC CAG 384

Thr Tyr Aria Leu Gfu Gin Ser Leu Thr Phe Gin Gin Ala leu Gin 115 120 125 AAG GAC ACC TCC GAC GTG CGG TGC ACC AAC GGC GAA CCG CCG GTT TTC 432 lys Asp Thr Ser Asp V Arg Arg Thr Asn Giy Giu Pro Pro Val Phe 130 135 140 GTC AAG CCG GAG AM GTA GTT GGA GTG ATT GGG GCT TCG GGG AGT TCG 480 112 ΡΕ1199359

Vaf Lys Pro Glu Lys Vat Go Gty Vsl te Giy Ata Ser Gly Ser Ser 145 150 155 160 QTC TCC .ATC ATO GTA GCC AAC ATO CTG AGG CTC TTC CAG ATC CCC CAG 528

(2) INFORMATION FOR SEQ ID NO: 10: (1) INFORMATION FOR SEQ ID NO: 10: (1) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 186 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Va! Girt tu Arg lys tu Tu Arg Arg Tu thy Thr tu Met tys Phe 1 5 10 15

Pro Cys Cys Vai Leu Glu Va! tu teu Cys Ala Leu Ala Aa Ala Ala 20 25 30

Arg Gly Gin Glu Met Tyr Ala Pro Hls Ser Ite Arg He Glu Gly Asp 35 40 45

Vaí Ttir tu Gly Gly Leu Phe Pro Vai Hls Ala Lys Gly Pro Ser Gly 113 ΡΕ1199359 SO 55 60 Vâi Pro Cys Gly Asp Ile Lys Arg Glu Asrt Gly Ile His Arg thy Giu 65 70 75 80

Ala Met Leu Tyr Ala tu Asp Gin lie Asn Ser Asp Pro Mn Leu Leu 85 90 95

Pro Asn Vat Thr Leu Gly Ata Arg fie Leu Asp Thr Cys Ser Arg Asp tOO 105 110

Thr Tyr Ala Leu Glu Gin Ser Leu Thr Phe Go Gin Ala Leu Ife Gin 115 120 125 tys Asp Thr Ser Asp Go Arg Cys Thr Asn Gly Glu Pro Pro Go Phe 130 135 140

Go Lys Pro Glu Lys Go Go Go Gly Go Go Gly Go Be Gly Ser Ser 145 150 155 160

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (i) SEQUENCE CHARACTERISTICS: (i) SEQUENCE CHARACTERISTICS: (i) SEQUENCE CHARACTERISTICS: (i) SEQUENCE CHARACTERISTICS: A) LENGTH: 2748 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: 114 ΡΕ1199359

(A) NAME / KEY: CDS (B) LOCATION: 1..2748 (D) OTHER INFORMATION: / product = " hmGluR7a " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATG GTC CAG CTG AGG AAG CTG CTC CGC GTC CTG ACT TTG AT0 AAG TTC 48

Met Vsi Gin Leu Arg lys Leu Lee Arg Vai Leu Thr Leu Met Lys Phe 1 δ 10 15

CCC TGC TGC GTG CTG GAG GTG CTC CTG TGC GCI CTG GCG GCG GCG GCG 96

Pro Cys Cys Vai Leu Giu Vai Leu Leu Cys Ata Leu Ala Ala Ala Ata 20 25 30, CGC GGC CAG GAG ATG TAC 6CC CCS CAC TCA ATC CGG ATC GAG GGG GAC 144

Arg Giy Gtn Gtu Met TyrAy Pro His Ser ile Arg lia Giu G and Asp 25 40 45 GTC ACC CTC GGG GGG CTG TTC CCC GTA CAC GCC AAG GGT CCC AGC GGA 192

(And 50 55 60 GTG CCC TGC GGC GAC ATC AAG AGG GAA AAC GGG ATC CAC AGG CTG GAA 240

VaSProCyaGiyAsptSeLysArgGiuAsnGfy He H & ArgLeuGlu 55 70 75 80 115 ΡΕ1199359

GCG ATG CTC TAC GCC CTG GAC CAG ATC AAC AGT GAT CCC MC CTA CTG 28 «

Ata Met leu Tyr Ata tew Asp Gin ile Aso Ser Aep Pm Aso leu Uu 85 SO 9 $

CCC AAC GTG ACG CTG GGC GCG CGO ATC CTG GAC ACT TGT TCC AGG GAC sm

Pm Asn Vat Thr leu Gly Ale Arg Ile Uu Asp Thr Cys Ser Arg Asp 100 108 110

ACT TAC GCG CTC GAA CAG TCG CTT ACT TTC GTC CAG GCG CTC ATC CAG 384

Thr Tyr Ala read Glu Gir »Ser leu Thr PhaVa! Oto Ala leu Gin 118 120 12 $ AAG GAC ACC TCC GAC GTG CGC TGC ACC MC GGC GM CCG CCG GTT TTC 432

Lys Asp Thr Ser Asp Vat Arg Cys Thr Asn Gly Glu Pro Pro Vat Phe 130 135 140

GTC MG CCG GAG AM GTA GTT © GA GTG ATT GGG GCT TCG GGG AGT TCG 480 will lys Pm Glu lys Vat Go Gly Go Gly Ata Ser Gly Ser Ser 145 150 155 180 GTC TCC ATC ATG GTA GCC GAC AAC ATC CTG AGG CTC TTC CAG ATC CCC CAG 528

Vai Ser lie Met Vat Ata Aso De leu Arg leu Pte Gin fe Pro Gtn 185 170 178 ATT AGT TAT GCA TCA ACG GOA CCC GAG CTA AGT GAT GAC CGG CGC TAT 576

Ite Ser Tyr Ala SerThr Ala Pm Glu le Ser Ser Asp Asp Arg Arg Tyr 180 185 190 116 ΔΕ1199359 GAC TTC TTC TCT CGC GTG GTG CCA CCC GAT ICC TTC CAA GCC CAG GCC 624

AspPhePfmSerArg Va! Go PrePreAsp Ser Phe Gin Ate Gli Ate 195 200 205 ATG GTA GAC ATT GTA MG GCC CTA GGG TGG MT TAT GTG TCT ACC CTC 672

MetVal Asplte Vai Lys Ala Leu Gly Trp Asn Tyr Will Be Thr Leu 210 21 $ 220 GCA TCG GM GGA AGT TAT GGA GAG AM GGT GTG GAG TCC TTC ACG CAG 720

AlaSerGtuGly SerTyrGly GIuLysGlyVatGluSerPfceThrGin 22 $ 230 235 240

ATT TCC AM GAG GCA GGT GGA CTC TGC ATT GCC CAG TCC GTG AGA ATC 758 Ito Ser Lys Giu Ata Gty GI Leu Cyaía Aia Gto Ser Vai Arg íle 245 250 255 CCC CAG GM CGC AAA GAC AGG ACC ATT GAC TTT GAT AGA ATT ATC AM 815 Pro Gin 01 "Arg Lys Asp Arg Thr He Asp PHe Asp Arg tto Lys 250 255 270 CAG CTC CTG GAC ACC CCC MC TCC AGG GCC GTC GTG ATT TTT GCC AAC 864

Gto Leu Leu Asp Thr Pro Asn Ser Arg Ala Will Go He PHe Ma Asn 275 280 285

GAT GAG GAT ATA MG CAG ATC CTT GCA GCA GCC AM AGA GCT GAC CAA 912

Asp Glu Asp Lys GSft He Leu Ma Ate Ato Lys Arg Ato Asp Gli »290 295 300 117 ΡΕ1199359

GTT GGC CAT TTT CTT TGG GTG GOA TCA GAC AGC TGG GGA TCC AAA ATA 9 »

Gly Hts Phe Lau Trp Go Gly Ser Asp Ser Trp Gly Ser Lys Be at 310 315 320 AAC CCA CTG CAC CAG CAT GAT GAT ATC GCA GAA GGG GCC ATC ACC ATT 1000

Asn Pra Leu Hls 6? HtO Glu Asp HO Ala Glu Gly Aia Ste Thrl 325 330 335 CAG CCG AAG CGA GCC ACG GTG GAA OGG TTT GAT GCC TAC TTT ACG TCC 1055

Glyn Pro lys Arg A Thr Va! Gtu Gly Phe Asp AfeTyrPha Thr Ser 340 345 350 CGT ACA CTT GAA AAC MC AGA AGA MT GTA TGG TTT GCC GAA TAC TGG 1104

Arg Thr leu Olu Asn Asn Ag Arç Asn Vai Τφ Pi »Ata Glu Tyr Τφ 355 380 365 GAG GAA AAC TTC AAC TGC MG TTG ACG ATT AGT GGG TCA AAA AAA GM 1182

Giu Gtu Asn Pha Asn CysLyslauTTr IJeSerGtySer lys lys Glu 370 375 380 GAC ACA GAT CGC AAA TGC ACA GGA CAG GAG AGA ATT GGA AM GAT TCC 1200

Asp Thr Asp Arg Lys Cys Thr Gly Gin Glu Arg As Gty lys Asp Ser 385 390 395 400 MC TAT GAG CAG GAG GGT AM GTC CAG TTC GTG ATT GAC GCA GTC TAT 1248

Asn Tyr Gíu Gin Gíu Gly lys Vai GIr Pha Vaií Asp Aia Vai Tyr 405 410 415 118 ΡΕ1199359

GCT ATS GCT CAC GCC CTT CAC CAC ATO AAC MO AT ATCTC TGT GCT GAC \ zm

AIAMtFaHIA Ala Leu Hls HSs Met Aso Lys Asp Leu Cj «Aia Asp 420 425 430 TAC COO GGT 6TC TGC CCA GAG ATS GAO CM GCT GGA CGC MG AAG TTG 1344

Tyr Arg Giy Vat Cys Pro Giu Met Gtu Gin Afa Gty Gty Lys Lys Uu 435 440 445 CTGMG TAT ATA CGC MT ÔTT MT TTC MT GGT AOT GCT GGC ACT CCA 1382

Leu Lys Tyr Ijb Arg Aso Vat Asn Phe Aso G! And Ser Afa G! And Utr Pro 450 455 450 GTO ATS TTT MC MG MC GGG GAT OCA CCT GGG CGT TAT GAC ATC TTT 1440

Go! Mel Phe Aso Lys Asn Gty Asp A! »Pro GiyA / g Tyr Asp Se Phe 455 470 475 450 CAG TAC CAG ACC ACA MC ACC AGC MC CCG GGT TAC CGT CTG ATC GGG 1458

Glr * Tyr Gin Thr Thr Aso Thr Ser Asn Pro Gly Tyr Arg Leu Se G ^ r 485 400 405 CAG TGG ACA GAC GM CTT CAG CTC MT ATA GM GAC ATG CAG TGG GGT 1535

Gtn Trp Thr Asp Gtu Leu Gin Leu Asn fle Gfu Asp Met Gin Trp Giy 500 505 510 AM GGA STC CGA GAG ATA CCC GCC TCA GTG TGC ACA CTA CCA TGT MG 1534

Lys Gfy Vat Aro Glu Ite Pro Aya Ser Vaf Cys Thr Leu Pro Cys Lys 515 820 523 119 ΡΕ1199359 CCA GGA CA © AGA AAG AAG ACA CA © AAA 6ÔA ACT CCT TGG TGT TÔG ACC 1832

Pm Giy Gin Arg Lys Lys Tfcr Gtn Lys GJy Thf Pm Cys Çys Ttp Thr 530 535 S40 TGT GAG CCT TGC GAT GGT TAC CAG TAC CAG TTT GAT GAG ATG ACA TGC 1680

Cys Giu Pm Cys Asp Giy Tyr Gin Tyr Glr * Pi »AspGlu Met Titr Cys 545 550 5S5 560

CAG CAT TGC CCC TAT GAC CA © AG © CCC AAT GM AAT CGA ACC GGA TGC 1728

Ghe Hys Cys Pm Tyr Asp Gin Arg Pr Ahi Glu As «Arg Thr Giy Cys 565 570 575 CA © GAT ATT CCC ATC ATC AAA CTG GAG TG © CAC TCC CCC TGG GCT GTG 1778

Gin Asp tys Prn Sie li lys leu Gtu Trp Hís Ser Pm Tm Afa Go 580 565 590

ATT CCT GTC TTC CTG GCA AT © GGG ATC ATT GCC ACC ATC TTT GTC 1824 IT Pro Go Phe lau Metalluu Giy ife tie Ate Thr ite Pi »Go 505 600 605 ATG ÔCC ACT TTC ATC CQC TAC AAT GAG ACG CCC ATT © TC C6 © GCA TCT 1872

Met AiaThr Phe Arg Arg Tyr Aso Asp Thr Profle V a Arg Ata Ser 610 615 620 GGG CGG GAA CTC ASC TAT © TT CTT TT © ACG GGC ATC TTT CTT TGC TAC 1920

Giy Arg Giu Leu Saf Tyr Go Leu leu Ti * Giy lie Ria Leu Cys Tyr 120 ΡΕ1199359 623 630 63 $ 640 ATC ATC ACT TTC CT ATO ATT GCC A CCA OAT OTO OCA OTO TGT TCT 1988 to Thr Phe thy Mel to Ala Lys Pnj Asp Go Ala Go Cys Ser 645 650 655 TTC COO CGA GTT TTC TTG GGC TTG GOT ATO TOC ATC AGT TAT OCA GCC 2016 phe ArgAtg Vaf PftateuGVteuGlyMetCys toSerTyrAiaAta 680 665 670 CTC TTG ACG AAA ACA AAT CGG ATT TAT CGC ATA TTT GAG CAG GGC MG 2064

Leu Leu Thr Lys Thr Asn Arg to Tyr Arg ile Phe GSu Gls Gly Lys §75 680 665

AM TCA GTA ACA GCT CCC AGA CTC ATA AGC CCA ACA TCA CAA CTG GCA 2112

Lys Ser Vai Thr Ala P »Arg Leu toSerPre Thr Ser Gin tu Ata« 90 695 700 ATC ACT TCC AGT TTA ATA TCA GTT CAG CTT CTA GGG GTG TTC ATT TG6 2190 lie Thr Ser Seriously Ser Vai Gin Leu Leu Gty Go Phe to Trp 705 710 715 720 TTT GGT GTT GAT CCA CCC MC ATC ATC ATA GAC TAC GAT 6AA CAC MG 2208 PheGly Go Asp Pro ProAsn He to to Asptyr AspGfuHisLys 725 730 735 ACA ATO MC CCT GAG CM GCC AGA GGG GTT CTC MG TGT GAC ATT ACA 2256

Thr Met Asn Pio Giu Gin Ala Arg Gty Vai leu Lys Cys Asp to Thr 740 745 750 121 ΡΕ1199359 GAT CTC CAA ATC ATT TGC TCC TTG GGA TAT AGC ATT CTT CTC ATG GTC 2304

Asp Leu Gin lie lie Cys Ser Leu Gly Tyr Ser lie Leu Leu Met Go 755 760 765 ACA TGT ACT GTG TAT GCC ATC AAG ACT CGG GGT GTA CCC GAG AAT TTT 2352

Thr Cys Thr Val Tyr Ala I Lys Thr Arg Gly Val Pro Glu Asn Phe 770 775 780 AAC GAA GCC AAG CCC ATT GGA TTC ACT ATG TAC ACG ACA TGT ATA GTA 2400

Asn Glu Ala Lys Prole Gly Phe Thr Met Tyr Thr Thr Cys Ile Val 785 790 795 800 TGG CTT GCC TTC ATT CCA ATT TTT TTT GGC ACC GCT CAA TCA GCG GAA 2448

Trp Leu Ala Phe Ile Pro Phe Phe Gly Thr Ala Gin Ser Ala Glu 805 810 815 AAG CTC TAC ATA CAA ACT ACC ACG CTT ACA ATC TCC ATG AAC CTA AGT 2496

Lys Leu Tyr lie Gin Thr Thr Thr Leu Thr ile Ser Met Asn Leu Ser 820 825 830 GCA TCA GTG GCG CTG GGG ATG CTA TAC ATG CCG AAA GTG TAC ATC ATC 2544

Ala Ser Goa Lea Gly Met Leu Tyr Met Pro Lys Goa Tyr Ile Ile 835 840 845 ATT TTC CAC CCT GAA CTC AAT GTC CAG AAA CGG AAG CGA AGC TTC AAG 2592

Ile Phe His Pro Glu Leu Asn Vai Gin Lys Arg Lys Arg Ser Phe Lys 850 855 860 GCG GTA GTC ACA GCA GCC ACC ATG TCA TCG AGG CTG TCA CAC AAA CCC 2640 122 ΡΕ1199359

Alava VatThr Wing ASaThfMeSer SerArg Uo Ser His Lys Pro 865 870 875 850 AGT GAC AGA CCC AAC GGT GAG GCA AAG ACC GAG CTC TGT GM MC GTA 2888

Asp Arg Pro Asn Gly Gtu Ala Lys Thr G) uu Cys GIu Asn Go 885 890 895 GAC CCA AAC AGC CCT GCT GCA AAA AAG MG TAT GTC AGT TAT MT MC 2738

Asp Pro Asn Ser Pro A ASa Lys Lys Lys Tyr Will Be Tyr Asn Asn 900 908 910 CTGGTTATCTA 2745

(A) LENGTH: 915 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein ( xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Go Gin Arg Arg Lys Leu Leu Arg Vsi Leu Thr tu Met Lys Phe 15 10 15

Pro 0¾¾ Cys Go Leu Gtu Go Leu Leu Cys Ala Leu Ala Ala Ala Ala 20 25 30 123 ΡΕ1199359

Arg Giy Gin Glu Met Tyr Ala Pro His Ser Its Arg ile Giu Giy Asp 35 40 45

Go Thr Leu Giy Giy Leu Phe Pro Go His Ala lys Giy Pro Ser Gty 50 55 60

Go Pro Cys Giy Asp Be Lys Arg Glu Asr * Giy He His Arg Leu Giu 65 70 75 80

Ala Mel Leu Tf Ala Leu Asp Gin He Asn Ser Asp Pro Asn Leu Leu 85 80 85

Pro Asn Vat Thr Leu Giy Aia Ag leu Asp Thr Cys Ser Arg Asp 100 105 110

Thr Tyr Ala Leu GSu Gin Ser Leu Thr Phe Gin Ais Leu Ile Gin 115 120 125

Lys Asp Thr Ser Asp Go Arg Cys Thr Mn Giy Glu Pro Pro Go Phe 130 135 140

Vai Lys Pro Glu Lys Vat Vai Giy Vai ile Giy Aía Ser Giy Ser Ser 145 150 155 160

Will Be Healthy Honey Will Go As Well Ile Leu Arg Leu Phe Gin Ite Pro Gin 165 170 175

Be Ser Tyr Ala Ser Thr Ala Pro Glu leu Ser Asp Asp Arg Arg Tyr 180 185 180

Asp Phe Phr Ser Arg Will Pro Pro Asp Ser Phe Gin Aκ Gfn Ala 195 200 205

Met Vat Asp ile Vai Lys Aia Leu Giy Trp Asn Tf Will Be Thr Leu 124 ΡΕ1199359 210 215 220

Ala Ser Giu Gly Ser Tyr Gly Glu Lys Gly V Glu Ser Pha Thr GSn 225 230 235 240 8 Ser Ly Glu Ala Gly Gty Leu Cys He Ala Gin Ser Vai Arg Ha 245 250 255

Pro Gin Giu Arg Lys Asp Arg Thr lie Mp Phe Asp Arg il Ba Lys 260 285 270

Gin Tu Leu Asp Thr Pro Asn Ser Arg Aia Goes To He Phe Ala Mn 275 280 285

Asp Glu Asp iie Lys Gin lie Leu Ata Ma Ala Lys Arg Ma Mp Gin 290 295 300

Go Gly Hys Phe Leu Trp Vat Gly Ser Asp Ser Trp Gly Ser Lys He 305 310 315 320

Mn Pro Leu His Gin His Glu Asp Be Ata Glu Gly Aia He Thr iie 325 330 335

Gin Pro Lys Arg Ala Thr Go Giu Gly Phe Asp Ala Tyr Phe Thr Ser 340 345 350

Arg Thr Leu Glu Asn Asn Arg Arg Asn Trp Phe Aia Glu Tyr Trp 355 360 365

Glu Glu Asn Phe Asn Cys Lys Leu Thr He Ser Gly Ser Lys Lys Giu 370 375 380

Asp Thr Mp Arg Lys Cys Thr Gly Gin Glu Arg He Gly Lys Mp Ser 365 390 395 400 125 ΡΕ1199359

Asn Tyr Giu Gin Giu Giy Lys Go Gin Gin »Go H® Asp Afa Go Tyr 405 410 415

Ala Met Ala Bis Ala Leu His His Met Asn Lys Asp Leu Cys A! A Asp 420 425 430

Tyr Arg Giy Vai Cys Pro Giu Met Glu G! N Aia Giy G! And Lys Lys Leu 435 440 445

Leu Lys Tyr Sie Arg Asn Go Asn Pha Asn Giy Ser Ala Giy Thr Pro 450 455 400

Go Met Phe Asn Lys Asn Giy Asp Aia Pro Giy Arg Tyr Asp ife Pt 465 470 475 480

Gin Tyr Gin Thr Thr Asn Thr Ser Asn Pro Giy Tyr Arg Leu lie Giy 485 430 495

Gin Trp Thr Asp Giu Leu Gin Leu Asn iie Giu Asp Met Gin Trp Giy 500 505 510

Lys Giy Go Arg Giu lie Pro Aia Be Va Cys Thr Leu Pro Cys Lys 515 520 525

Pro Giy Gin Arg Lys Lys Thr Gin Lys Giy Thr Pro Cys Cys Trp Thr §30 535 540

Cys Glu Pro Cys Asp Giy Tyr Gin Tyr Gtn Phe Asp Giu Met Thr Cys 545 550 555 560

Gin His Cys Pro Tyr Asp Gin Arg Pro Asn Glu Asn Arg Thr Giy Cys 565 570 575 126 ΡΕ1199359

Gin Asp Ite Lys Leu GI Trp Hys Ser Pr »Trp Ala Go 580 585 590 Pro Pro Phe Leu Ata Met Leu Giy liete Ata Thr fo Phe Go 535 800 805

Honey Wing Thr Pha te Arg Tyr Aso Asp Thr Pm te Vat Arg Wing Ser StO 615 820

Giy Arg Giu Leu Ser Tyr Go Leu Leu Thr Gly tie Phe Leu Cys Tyr 625 630 638 840 liete Thr Pha Leu Met te Ala Lys Pro Asp Goa Vat Cys Ser 845 650 855

Phe Arg Arg Vat Phe Leu Gty Leu Gly Mel Cys Ser Tyr Ata Ate »860 865 670

Leu Leu Thr Lys Thr Asn Arg ite Tyr Arg fie Phe Glu Gin Gty Lys 675 680 885

Lys Ser Vat Thr Ala Pro Arg Leu ser Pro Thr Ser Gin leu Ala 690 895 700 te Thr Ser Ser Leu tte Ser Vat Gtn Leu Leu Gty Vat Phe te Trp 705 710 715 720

Phe Giy Vaf Asp Pro Pro Acid Asp Tyr Asp Glu HSs Lys 725 730 735

Thr Met Asn Pro Giu Gin Ala Arg Giy Vai leu Lys Cys Asp te Thr 740 745 750

Asp Leu Gin Ite Cys Ser Leu Gly Tyr Ser lie Leu Leu Met Vat 127 ΡΕ1199359 755 760 765

Thr Cys Thr Va! Tyr Ala tysThrArg Gty Vai Pr »Glu Asn Phe 770 775 750

Mn Glu Ata Lys Pro te Gly Phe TTtr Met Tyr Thr Thr Cys lie Val 765 790 795 600

Trp Leu Afa Phe Ile Pro Phe Phe Gty Thr Ata Gin Ser Aia Gfu 805 810 815

Lys Leu Tyr ile Gin Thr Thr Thr Leu Thr lie Ser Mel Asn Leu Ser 820 825 830 Aia Ser Vai Aia Leu Giy Met Leu Tyr Met Pro Lys Will Tyr tte He 835 840 845 te Phe Hls Pro GSu thy Asn Go Gin Lys Mg Lys Arg Ser Phe Lys 850 855 860

Ata Vai Vai Thr Aia Aia Thr Met Ser Ser Arg Arg Leu Ser Hls Lys Pro 865 870 875 880

Ser Asp Arg Pro Asn Giy Gfu Aria Lys Thr Glu Leu Cys Glu Asn Go 885 890 895

Asp Pro Asn Ser Pro Aia Ala Lys Lys Lys Tyr Will Be Tyr Asn Asn 900 905 910

Leu Va! SEQUENCE CHARACTERISTICS: (A) LENGTH: 2769 base pairs 128 ΡΕ1199359 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 1..2769 (D) OTHER INFORMATION: / product = " hmGluR7b " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ATG GTC CA © CTOT AO © AAS CT © CTC CGC © TC CTG ACT TTG ATS AA © TTC 48

MetValGinteuArgLysLeuLeuAfgVaí Leu Thr Leu Mat Lys Pfte 1 S 10 15

CCC TGC TGC TG TGG © A TGG TG CTC TG TGC TG GCG GC GC GC GC GC

Fro Cys Cys Va »Leu Glu Vai Leu Leu Cys Aia Leu AlaAia Aia Ata 20 25 30 CSC GGC CA © GA © AT © TAC GCC CCG CAC TCA ATC CGG ATC GA © GGG GAC 144

Arg Gyy Gin Gyu Met Tyr Aia Pm His Ser !! and Arg © asp 35 40 45 GTC ACC CTC © GO © 0 © CTG TTC CCC © TA CAC GCC AA © GGT CCC AGC © GA 192

VaITfcfLeuGlyGiyLewPJwPravaiHisAlalysGfyFfoSefGly 50 55 80 129 ΡΕ1199359 GTG CCC TGC GGC GAC ATC AAG AGG GAA AAC GGG ATC CAC AGG CTG GAA 240

Go Pro Cys Gly Asp lys Arg Glu Asn Gly lys His Arg Leu Glu 65 70 75 80 GCG ATG CTC TAC GCC CTG GAC CAG ATC AAC AGT GAT CCC AAC CTA CTG 288

Ala Met Leu Tyr Ala Leu Asp Asn Asp Ser Asp Pro Asn Leu Leu 85 90 95 CCC AAC GTG ACG CTG GGC GCG CGG ATC CTG GAC ACT TGT TCC AGG GAC 338

Pro Asn Go Thr Leu Gly Ala Arg lie Leu Asp Thr Cys Ser Arg Asp 100 105 110 ACT TAC GCG CTC GAA CAG TCG CTT ACT TTC GTC CAG GCG CTC ATC CAG 384

Thr Tyr Ala Leu Glu Gin Ser Leu Thr Phe Go Gin Ala Leu lie Gin 115 120 125 AAG GAC ACC TCC GAC GTG CGC TGC ACC AAC GGC GAA CCG CCG GTT TTC 432

Lys Asp Thr Ser Asp V Arg Arg Cys Thr Asn Gly Glu Pro Pro Val Phe 130 135 140 GTC AAG CCG GAG AAA GTA GTT GGA GTG ATT GGG GCT TCG GGG AGT TCG 480

Go Lys Pro Glu Lys Go Go Gly Go Gly Ala Ser Gly Ser Ser 145 150 155 160 GTC TCC ATC ATG GTA GCC AAC ATC CTG AGG CTC TTC CAG ATC CCC CAG 528

It will be Met Met Val Ala Asn He Leu Arg Leu Phe Gin lie Pro Gin 130 ΡΕ1199359 165 170 175 ATT AGT TAT GGA TCA ÂCG OCA CCC OAO CTA AGT GAT GAC COO CGC TAT 576 Ile Set Tyr Ate Ser Thr Ala Pro Glu Leu Ser Asp Asp Arg Arg Tyr 180 186 180 gac rrc ttc tct cgc gtg gtg cca ccc gat tcc rrc caa gcc cag gcc 624

Asp Phe Phe Ser Arg Will Pro Pro Asp Ser Re Gin Gin 185 2θO 205 ATG GTA GAC ATT GTAAAG GCC GTA GGC TGG AAT TAT GTG TCT ACC CTC 672

Met Go Asp Ha Go Lys Ate Leu Gly Trp Asn Tyr Will Be Thr Leu 210 215 220 GGA TCG GAA GGA AGT TAT GGA GGA AAA GGT GTG GAG TCC TTC ACG CAG 720

Ate Ser Glu Gly Ser Tyr Gly GSu Lys Gly Go Glu Ser Phe Thr Gin 225 230 235 240 ATT TCC AAA GAG GCA GGT GGA CTC TGC ATT GCC CAG TCC GTG AGA ATC 788 Ile Ser Lys Glu Ate Gly Gly Leu Cys Ite Ate Gin Ser VIA Arg 245 250 255 CCC CAG GAA CGC AAA GAC AGG ACC ATT GAC TTT GAT AGA ATT ATC AAA 816

Pio Gin Glu Arg Lys Asp Arg Thr tie Asp Rhe Asp Aig ite tte Lys 280 265 270

CAG CTC CTG GAC ACC CCC AAC TCC AGG GCC GTC GTG ATT TTT GCC AAC 884

Gin Leu Leu Asp Thr Pro Asn Ser Arg Ala Vai lie Phe Ale Asn 275 280 285 131 ΡΕ1199359

GAT GAG GAT ATA AAG CAG ATC CTT GCA GCA GCC AAA AGA GCT GAC CAA 912

Asp Glu Asp lie Lys Gin tis Leu Ala Ala Ala tys Arg Ala Asp Gin 290 285 300 GTT GGC CAT ΠΤ CTT TGG GTG GGA TCA GAC AGC TGG GGA TCC AAA ATA 9β0

Go Gly His Phe Leu Trp Go Gly Ser Asp Ser Trp Gly Ser Thys 305 310 315 320

AAC CCA CTG CAC CA © CAT GAA GAT ATC GCA GAA GGG GCC ATC AGC ATT 1008

Asn Pt Leu Hfs Gin Hts Glu Asp fie Ala Glu Gly Ala tte Thr lia 325 330 335 CAG CCC AAG CGA GCC ACG GTG GAA GGG TTT GAT GCC TAC TTT ACG TCC 1055

Gin Pro Lys Afg Ate Thr Go Glu Gly PI »Asp Ala Tyr Phe Thr Ser 340 345 350

CGT ACA CTT GAA AAC AAC AGA AGA AAT GTA TGG TTT GCC GAA TAC TGG 1104

Afg Thr Leu Glu Asn Asn Arg Arg Asn Trp Phe Ate Glu Tyr Τη »355 380 3Θ5

GAG GM AAC TTC AAC TGC AAG TTG ACG ATT AGT GGG TCA AAA AAA GAA 1152

Glu Glu Asn Phe Asn Cys Lys Leu Thr be Ser Gly Ser Lys Lys Glu 370 375 380

GAC ACA GAT CGC AAA TGC ACA GGA CAG GAG AGA ATT GGA AAA GAT TCC 1200

Asp Thr Asp Arg Lys Cys Thr Gly Gin G Arg Arg Gly Lys Asp Ser 132 ΡΕ1199359 385 3SQ 305 400

AAC TAT GAG GAG GAG 6GT AAA GTC CAG TTC GTG ATT GAC GCA GTC TAT 1245

Aso Tyr Glu Gin Glu Gly Lys Go Gin Pfte Vat lie Aap Ala Vai Tyr 405 410 415

GCT ATG GCT CAC GCC CTT GAC CAC ATG AAC AAG GAT CTC TGT GCT GAC 1208

AlaMet AfaHisAllHisHisMelAsnLysiAspLÉmCysAlaAsp 420 425 430 TAC CGG GGT GTC TGG CCA GAG ATG GAG CAA GCT GGA GGC AAG AAG TT3 1344

Tyr Arg Gly V Cys Pm Glu Uet Glu Gin Ala Gly Gly Lys Lys Leu 435 440 445 CTG AAG TAT ATA CGC AATGTTAAT TTC AAT GGT AGT GCT GGC ACT CCA 1392 Leu Lys Tyr De Arg Asn Vaf Asn PI »Asn Gly Ser Ala Gly Thr Pro 450 455 460 GTG ATG TTT AAC AAG AAC GGG GAT GCA CCT GGG CGT TAT GAC ATC TTT 1440

Go to Met Pt »Asn Lys Asn Gly Asp Ala Pro Gly Arg Tyr Asp tte Phe 465 470 475 480

CAG TAC CAG ACC ACA AAC ACC AGC AAC CCG GGT TAC CGT CTG ATC GGG 1488

Gin Tyr Gin Thr Thr Asn Thr Ser Asn Pro Gty Tyr Arg Leu ile Gly 485 490 495 CAG TGG ACA GAC GAA CTT CAG CTC AAT ATA GAA GAC ATG CAG TGG GGT 1538

Gin Trq Thr Asp Glu Leu Gin Leu Aso Ite Glu Asp Met Gin Trp © ly 133 ΡΕ1199359 500 505 510 AM GGA GTC CGA GAG ATA OCC GCC TCA GT6 TGO ACA CTA CCA TGT MG 1584

Lys GfyVatAigGtuito Pro Ata SerVa? Cys Thr Leu Pro Cys Lys 515 520 525

CCA GGA CAG AGA MG AAG ACA CAG AAA GGA ACT CCT TGC TGT TGG ACC 1832

Pro Gly Gin Arg Lys Lys Thr Gin Lys Gly Thr Pm Cys Cys Trp Thr 530 535 540 TGT GAG CCT TGC GAT GGT TAC CAG TAC CAG TFT GAT GAG ATG ACA TGC 1680

Cys Gtu Pro Cys Asp Gly Tyr Gin Tysr Gluth Phe Asp Glu Met Thr Cys 545 550 555 560 CAG CAT TGC CCC TAT 6AC CAG AGG CCC MT GM MT CGA ACC GGA TGC 1728

Gin His Cys Pro Tyr Asp Gin Arg Pro Asn Glu Asn Arg Thr Gly Cys 565 570 575 CAG GAT ATT CCC ATC ATC AM CTG GAG TGG CAC TCC CCC TGG GCT GTG 1776

Gin Asp ls Pro tis lie Lys Leu Giu Trp His Ser Pro Trp AIS Go 580 585 $ 30

ATT ATC ATC ATC ATC ATC TTT GTC 1854 IT Pro Go Phe Leu Ais Met Leu Glyte Ite Ata Thr fo Phe Val 595 600 805

ATG GCC ACT TTC ATC CGC TAC MT GAC ACG CCC ATT GTC CGG GCA TCT 1872 134 ΡΕ1199359 MSet Ala Thr Phe tle Aig Tyr Mn Asp Thr Pm te Va! Arg Ala Ser 61Q 615 620 GGG CGG GAA CTC AGC TAT GTT CTT TTG ACG GGC ATC TTT CTT TGC TAC 1920

GlyArg Giu Leu Ser Tyr Go Leu Leu Thr Gly te Phe Lee Cys Tyr 625 636 635 840 ATC ATC ACT TTC CTG ATG ATT GCC AAA CCA GAT GTG GCA GTG ΤΘΤ TCT 1968

Tetracycline Amino Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid Acid

Phe Arg Arg Val Phe Leu Gly Leu Gly Met Cys and Ser Tyr Ala Ala 666. 665 670

CTC TTG ACG AAA ACA AAT CGG ATT TAT CGC ATA TTT CAG CAG GGC AAG 2064

Leu Leu Thr Lys Thr Aso Arg Ife Tyr Arg te Phe Giu Gin Gly Lys 676 660 665 AAA TCA GTA ACA GCT CCC AGA CTC ACTA AGC ACA ACA CAA CTG GCA 2112

Lys Ser Vai Thr Aia Pro Arg Lm te Ser Pro Thr Ser Gin leu Ala 690 695 700 ATC ACT TCC AGT TTA ATA TCA GTT CAG CTT GTA GGG GTG TTC ATT TGG 2160 te Thr Ser Ser Leu te Ser Goa Gta Leu Leu Giy Go Phe te Trp 705 710 715 720 TTT GGT GTT GAT CCA CCC AAC ATC ATC ATA GAC TAC GAT GAA CAC AAG 2206 Phe Gly Go Asp Pro Pro Asn AspTyr AspQuHis Lys 725 730 735 135 ΔΕ1199359 ACA ATG AAG CCT GAG CAA GCC AGA GGG GTT CTC AAG TGT GAC ATT ACA 2258

ThrMeSIn Pro Glu Gin Ala Arg Gly Go Leu Lys Cys Asp tie Thr 740 745 750 GAT CTC CAA ATC ATT TGC TCC TTG GGA TAT AGC ATT CTT CTC ATG GTC 2304

Asp Leu Gin la Cys Ser Leu Gly Tyr Ser le leu Leu Met Go 755 750 765

ACA TGT ACT GTG TAT GCC ATC AAG ACT CGG GGT GTA CCC GAG AAT TTT 2352

Thr Cys Thr Val Tyr Ala lys Thr Arg GfyVal Pio Glu Asn Ph® 770 775 780 AAC GAA GCC AAG CCC ATT GGA TTC ACT ATG TAC ACG ACA TGT ATA GTA 2400

Asn Glu Ala Lys Gly Ph Thr Mel Tyr Thr Thr Cys He Go 785 790 795 800 TGG CTT GCC TTC ATT CCA ATT TTT TTT GGC ACC GCT CAA TC GCA GAA 2448 Trp Leu Ala Ph s te Pro te Phe Phe Gly Thr Ala Gin Be Ala Glu to the A ctions MG CTC TAC ATA CAA ACT ACC ACG CTT ACA ATC TCC ATG MC CTA AGT 2496 lys Leu Tyr Thr Thr Thr Leu Thr Ser Met Asn Leu Ser 820 825 830 GCA TCA GTG GCG CTG GGG ATG CTA TAC ATG CCG AAA GTG TAC ATC ATC 2544

Ala Ser Vai Leu G! And Met Leu Tyr Met Pr® Lys Vai Tyr te 835 840 845 136 ΡΕ1199359 ATT TTC CAC CCT GAA CTC AAT GTC CAG A CGG AAG CGA AGC TTC AAG 2592 iiePheHfsProGIUteuAsnVaiGinLysArgLysAIGSerPheLys 850 855 860 GCG GTA GTC ACA GCA GCC ACC ATG TCÀ TCG AGG CTG TC A CAC AAA CCC 2640

Ala Vai Vai Thr Ala Ala Thr Mbi Ser Ser Afg Leu Ser Hls Lys Pro 865 870 875 880 AGT GAC AGA CCC AAC GGT GAG GCA AAG ACC GAG CTC TGT GAA MC GTA 2688

Ser Asp Arg Pro Asn GSy GJu Ala Lys Thr Giu Leu Cys Gfu Asn Go 885 890 895

GAC OCA MC MC TGT ATA CGA CCA GTA AGA MG AGT GTA CM MG TCT 2736

# Pro Asean Cys lie Pro Pro Go Arg Lys Ser Vai Gin Lys Ser SOO SOS 910 GTT ACT TGG TAC ACT ATC CCA ACA GTA TA 2763

SEQUENCE CHARACTERISTICS: (A) LENGTH: 922 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (T) ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 137 ΡΕ1199359

Met Vai Gin Leu Arg Lys Leu Leu Arg Leu Thr Leu Met Lys Phe 15 10 15

Pro Cys Cys Vai Leu Glu Vai Leu Leu Cys Ala Leu Ala Ala Ala Ala 20 25 30

Arg Gly Gin Glu Met Tyr Ala Pro His Ser Ile Arg Ile Glu Gly Asp 35 40 45

Go Thr Leu Gly Gly Leu Phe Pro Go His Wing Lys Gly Pro Ser Gly 50 55 60

Go Pro Cys Gly Asp lie Lys Arg Glu Asn Gly lie His Arg Leu Glu 65 70 75 80

Ala Met Leu Tyr Ala Leu Asp Gin lie Asn Ser Asp Pro Asn Leu Leu 85 90 95

Pro Asn Go Thr Leu Gly Ala Arg lie Leu Asp Thr Cys Ser Arg Asp 100 105 110

Thr Tyr Ala Leu Glu Gin Ser Leu Thr Phe Go Gin Ala Leu lie Gin 115 120 125

Lys Asp Thr Ser Asp Go Arg Cys Thr Asn Gly Glu Pro Pro Go Phe 130 135 140

Go Lys Pro Glu Lys Go Go Goly Go Go Goly Go Be Gly Ser Ser 145 150 155 160

It will be Met Met Val Ala Asn lie Leu Arg Leu Phe Gin lie Pro Gin 170 175 165 138 ΡΕ1199359 lie Ser Tyr Ala Ser Thr Ala Pro Glu Leu Ser Asp Asp Arg Arg Tyr 180 185 190

Asp Phe Phe Ser Arg Will Pro Pro Asp Ser Phe Gin Ala Gin Ala 195 200 205

Met Val Asp lie Val Lys Ala Leu Gly Trp Asn Tyr Will Be Thr Leu 210 215 220

Ala Ser Glu Gly Ser Tyr Gly Glu Lys Gly Go Glu Ser Phe Thr Gin 225 230 235 240 lie Ser Lys Glu Ala Gly Gly Leu Cys lie Ala Gin Ser Vai Arg He 245 250 255

Pro Gin Glu Arg Lys Asp Arg Thr Ile Asp Phe Asp Arg Ile He Lys 260 265 270

Gin Leu Leu Asp Thr Pro Asn Ser Arg Ala It Will Go Phe Ala Asn 275 280 285

Asp Glu Asp lie Lys Gin lie Leu Ala Ala Ala Lys Arg Ala Asp Gin 290 295 300

Gly His Phe Leu Trp Gly Gly Ser Asp Ser Trp Gly Ser Lys lie 305 310 315 320 i Uim Uto ΛΙι A Ι ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΐΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ ΠΙ I III 11 '325 330 335

Gin Pro Lys Arg Ala Thr Go Glu Gly Phe Asp Ala Tyr Phe Thr Ser 340 345 350

Arg Thr Leu Glu Asn Asn Arg Arg Asn Trp Phe Ala Glu Tyr Trp 139 ΡΕ1199359

Cys Glu Pro Cys Asp Gly Tyr Gin Tyr Gin Phe Asp Glu Met Thr Cys 545 550 555 560

Gin His Cys Pro Tyr Asp Gin Arg Pro Asn Glu Asn Arg Thr Gly Cys 565 570 575

Gin Asp lie Pro-Ile Lys Leu Glu Trp His Ser Pro Trp Ala Val 560 585 590 Ile Pro Val Phe Leu Ala Met Leu Gly Ile Ile Ala Thr Ile Phe Val 595 600 605

Met Ala Thr Phe Ile Arg Tyr Asn Asp Thr Pro Ile Val Al Arg Ser 610 615 620

Gly Arg Glu Leu Ser Tyr Go Leu Leu Thr Gly lie Phe Leu Cys Tyr 625 630 635 640 Ile lie Thr Phe Leu Met lie Ala Lys Pro Asp Goa Ala Goa Cys Ser 645 650 655

Phe Arg Arg Val Phe Leu Gly Leu Gly Met Cys Ile Ser Tyr Ala Ala 660 665 670

Leu Leu Thr Lys Thr Asn Arg Ile Tyr Arg Ile Phe Glu Gin Gly Lys 675 680 685

Lys Ser Vai Thr Ala Pro Arg Leu lie Ser Pro Thr Ser Gin Leu Ala 690 695 700 lie Thr Ser Ser Leu lie Ser Vai Gin Leu Leu Gly Vai Phe lie Trp 705 710 715 720 140 ΡΕ1199359

Phe Gly Va! Asp Pm Pro Asn Ite He & Asp Tyr Asp Glu His Lys 725 730 735

Thr Môí Asn Pm Gíu Gtrt Ala Arg Giy Va! Leu Lys Cys Asp Thr 740 745 750

Asp Leu Gin tfe Jte Cys Ser Leu Giy Tyr Ser lie Leu Leu Mel VeJ 755 760 765

Thr Cys Thr VaJ Tyr AM «and Lys Thr Arg Giy Val Pm Glu Asn Phe 770 775 780

Asn Glu Ays Lys Pro He Giy Phe Thr Met Tyr Thr Thr Cys tb Go 785 790 795 800

Trp Leu Ala Phe Iie Fm Ite Phe Phe Gly Thr Ala Gin Ser Ala Glu 805 810 815

Lys Leu Tyr Jte Gin Thr Thr Thr Leu Thr lie Ser Me! Asn Leu Ser 820 825 830 AM Ser Vai Ala Leu Giy Me! Leu Tyr Met Pro Lys Vaf Tyr Ile 835 840 845 Ile Phe His Pm Glu Leu Asn VaJ Gin Lys Arg Lys Arg Ser Ph® Lys 850 855 880

Ala vai va Thr Ala Aia Thr Mat Ser Ser Arg Arg Leu Ser His Lys Pro 865 870 875 880

Ser Asp Arg Pm Asn Gly Glu Ata Lys Thr Glu Leu Cys Glu Asn Go 335 @ 90 895

Asp Pro Asn Asn Cys He Pro Pro VaJ Arg Lys Ser Vai Gin Lys Ser 141 ΡΕ1199359 900 905 910

(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 630 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 1..630 (D) OTHER INFORMATION: / product = " hmGluR6 partial " (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GTG GAG GCC CTG CAG TGG TCT GGC GAC CCC CAC GAG GTG CCC TCG TCT 43

Go Glu Ala Leu Gin Ttp Ser Gty Asp Pro His Giu Go Pro Ser Ser 15 10 15 CTG TGC AGC CTG CCC TGC GGG CCG 6GG GAG CGG AAG AAG ATG GTG AAG 96

LeuCysSerLetiProCysGlyProcayGluArgLysLysMetValLys 20 25 30 GGC GTC CCC TGC TGT TGG CAC TGC GAG GCC TGT GAC GGG TAC CGC TTC 144 142 ΡΕ1199359 GÎY Vai Pro Cys Cys Trp His Cys Glu Ala Cys Asp Giy Tyr Arg Phe 35 40 45 CAG GTG GAC GAG TTC ACA TGC GAG GCC TGT CCT GGG TAC ATG AGG CCC 1Θ2

Gin Go Asp Gfu Pfte Thr Cys Glu Afa Cys Pro Gty Tyr Met Arg Pro 50 55 50 ACN CCC AAC CAC ATG NNA CTT NNG CCC ACA CCT GTG GTG CGC CTG AGG 240

Xaa Pro Asn His Ile Xaa Leu Xaa Pro Thr Pro Vaf Vai Arg Leu Ser 55 70 75 80 TGG TGC TCC CCC TGG GCA GCC CCS CCG CTC CTC CTG GCC GTG CTG GGC 288

Trp Ser Ser Pro Trp Ate Aia Pro Pro Uu Leu tu Aia Vai leu GSy · 35 90 55

ATC GTG GCC ACT ACC ACG GTG GTG GCC ACC TTC GTG CGG TAC AAC MC 336 ile Go Ala Thr Τι »Thr Go Go Ala TI» Pfte Go Arg Tyr Aso Asn 100 105 110 ACG CCC ATC GTC CGG GCC TCG GGC CGA GAG CTC AGC TAC GTG CTC CTC 364

Thr Prola Vai Arg Afa Ser Gty Arg Giu Leu Ser Tyr Va! Leu Leu 115 120 125 ACC GGC ATC TTC CTC ATC TAC GCC ATC ACC TTC CTC ATG GTG GCT GAG 432

Thr Gty Be Phe Leu Ile Tyr Aja tta Thr Phe Leu Met Go Ala Gtu 130 135 140 143 ΡΕ1199359 CCT GGG GGA GCG GTC TGT GCC GCC CGC AGG CIC TTC CTG GGC CTG GGC 460

Pro Gly Ate Ala Go Cys Ate Arg Arg Leu Phe Leu Gly Leu Giy 146 160 155 160 ACG ACC CTC AGC TAC TCT GCC CTG CTC ACC AAG ACC AAC CGT ATC TAC 528

Thr Thr Leu SerTyr Ser Ala Leu Leu Thr Lys Thr Asrt Aig tte Tyr 165 170 175 CGC ATC ITT GAG CAG GGC AAG CGC TCG GTC ACA CCC CCT CCC TTC ATC 578

Arg tle Pite Glu Gin Gty Lys Arg Ser Go Pro Th Pro Pro Pro Phe tte 180 185 190

AGC CCC ACC TCA CAG CTG GTC ATC ACC TTC AGC CTC ACC TCG CTG CAG 624

Ser Pro Thr Ser Gin Leu Vai Ite Thr Phe Ser Leu Thr Ser Leu Gin 195 200 205 GTG GGG 630

VatGly 210 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 210 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: SEQUENCE DESCRIPTION: SEQ ID NO: 16: 144 ΡΕ1199359

Go m Ala leu Gin Trp Be Gly Asp Pro Hís Gtu Go Pro Be Ser t S 10 15

Leu Cys Ser Leu Pro Cys Gly Pro Oty Glu Arg Lys Lys Met Val Lys 20 25 30 Gly and Pro Pro Cys Cys Trp His Cys Gly Afa Cys Asp Gty Tyr Arg Phe 35 40 45

Gin Go Asp Glu Phe Thr Cys Glu Ata Cys Pro Gly Tyr MatArg Pro §0 55 SO

Xaa Pro Asn Hls He Xaa tu Xaa Pm Thr Pro Will Go Arg Leu Ser 65 70 75 80

Trp Ser Ser Pro Trp Ala Ala Pro Pro leu Leu Leu Ala Go Leu Gly 85 m 95 ire Go Ala Thr Thr Thr VaI Go Ala Thr Phe Go Arg Tyr Asn Aso 100 105 110

Thr Pm He Go Arg Ala Gly Arg Glu Leu Ser Tyr Vat Leu Leu 115 120 125

Thr Gly He Phe Leu He Tyr Ala Thr Phe Leu Mel Going Ala GSu 130 135 140

Pro Gly AJa Ala Vai Cys Ala Ala Arg Arg Leu Phe Leu Gly Leu Gly 145 150 155 180

Thr Thr thy Ser Tyr Ser Ala Leu Leu Thr Lys Thr Mn Arg Ele Tyr 185 170 175 ΡΕ1199359 145

Arg Ke Phe G! U Gin Oy Lys U $ Be Go Thr For Pro Pra Phe te 180 136 190

Ser Pro Tbr Ser Gin Leu Go Ste tle Phe Ser Leu 1% Ser tu Gin 195 200 205

ValGIy 210

Lisbon, November 10, 2011

Claims (28)

A purified human metabotropic glutamate (hmGluR) receptor comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO: 16. 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 metabotropic glutamate receptor that is encoded by a nucleotide sequence capable of hybridizing under high stringency conditions of SEQ ID NO: 1 or 15 and which the polypeptide product shows under conventional condition in the radioimmunoassay of cAMP which AP-4 has an agonistic effect at a concentration of less than 1 mM. 4. 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 comprising a promoter and a DNA coding for this receptor whose DNA is controlled by said promoter. 5. Use of a receptor according to claim 1, 2, or 3 for screening a compound that modulates the activity of said receptor. An isolated nucleic acid comprising a nucleic acid encoding a receptor according to claim 1, 2, or 3. The nucleic acid added according to claim 6 which is a DNA 8. The DNA of claim 7 selected from the group consisting of the DNAs having the nucleotide sequence set forth in SEQ ID NOs. 1 or 15, respectively. A process for the preparation of an added nucleic acid according to claim 7 comprising chemical synthesis, recombinant DNA technology or polymerase chain reaction (PCR). A DNA according to claim 7 which is a hybrid vector. A host cell comprising a DNA of claim 10. A eukaryotic host cell expressing the DNA of claim 10. 3 ΡΕ1199359 A host cell according to claim 11 or 12 which is a mammalian cell. 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. A process for the preparation of a host cell according to claim 11 comprising transfecting or transforming the host cell with the hybrid vector of claim 11. A purified mRNA complementary to the DNA of claim 8. A method for identifying the DNA encoding a hmGluR subtype according to claim 1, 2, or 3 comprising contacting human DNA with a probe, and identifying DNA (s) that hybridize to said probe, wherein said probe is a nucleic acid probe comprising at least 14 contiguous bases of the nucleic acid according to claim 6 or 7, or its complement and which specifically binds to any nucleic acid of claim 6 or 7, or its complement, conditions of high stringency. 4 ΡΕ1199359 A method for identifying compounds which bind to a hmGluR subtype comprising the use of a receptor protein according to claim 1, 2, or 3 in a competition binding assay. An assay for identifying compounds that modulate the activity of an hmGluR subtype according to claim 1, 2, or 3 comprising contacting the cells of claim 12 with at least one compound or signal the ability to modulate the activity of said subtype of receptor is sought, and subsequently - to analyze the cells for a difference in functional response attributable to said receptor. An assay according to claim 19 comprising contacting the cells or claim 12 with at least one compound or signal whose ability to modulate the second messenger activity of a recipient subtype of the invention has been sought to determine, and subsequently - monitor said cells to a change in the level of a particular second messenger. A method for detecting a glutamate agonist or an allosteric modulator of a hmGluR subtype of 5 ΡΕ1199359 according to claim 1, 2, or 3 having agonistic activity comprising the steps of (a) exposing a compound to a hmGluR subtype of according to claim 1, 2 or 3 coupled to a response pathway under conditions and for a time sufficient to allow interaction of the compound with the receptor and a pathway-associated response, and (b) detecting an increase or decrease on stimulation of the response pathway resulting from the interaction of the compound with the hmGluR subtype relative to the absence of the compound tested and thereby determining the presence of an allosteric agonist or modulator having agonist-like activity. A method for identifying a glutamate antagonist or an allosteric modulator of an hmGluR subtype 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 for a hmGluR subtype according to claim 1, 2, or 3 in a response pathway under conditions and for a time sufficient to allow interaction of the agonist with the receptor and a pathway-associated response, and (b) detecting an inhibition of agonist response pathway stimulation resulting from interaction of the test compound with the hmGluR subtype, relative to stimulation of the response pathway induced by the isolated glutamate agonist, and thereby determining the presence of a glutamate antagonist or a allelic modulator having antagonist-like activity. 6 ΡΕ1199359 An antibody that specifically binds to a protein of claim 1, 2, or 3. An antibody according to claim 23 which is a polyclonal antibody. An antibody according to claim 23 which is a monoclonal antibody. A method for modulating the signal transduction activity of an hmGluR subtype according to claim 1, 2, or 3 comprising contacting said receptor with an antibody of claim 24. A receptor according to claim 1, 2, or 3 obtainable by recombinant DNA technology. A fusion protein comprising a receptor according to claim 1, 2, or 3.