CA2343574A1

CA2343574A1 - Metabotropic gaba receptor complex issued from the central nervous system

Info

Publication number: CA2343574A1
Application number: CA002343574A
Authority: CA
Inventors: Hans-Christian Kornau; Gisela Eisenhardt; Rohini Kuner; Kirsten Hirschfeld
Original assignee: Individual
Current assignee: Sygnis Pharma AG
Priority date: 1998-09-14
Filing date: 1999-09-11
Publication date: 2000-03-23
Also published as: EP1112363A1; WO2000015786A1; JP2002525052A; AU6081799A

Abstract

The invention relates to isolated proteins which code for metabotropic GABA receptors and which, together with GABAB receptor proteins, form a metabotropic GABA receptor complex (= protein heteromer). The invention also relates to nucleic acid sequences or recombinant nucleic acid constructs whi ch code for the proteins. In addition, the invention relates to host organisms or transgenic animals containing the nucleic acid sequences or the recombinant nucleic acid constructs and to monoclonal or polyclonal antibodies which are directed against the isolated proteins. The invention also relates to a meth od for locating substances with a specific affinity to binding to the inventive novel GABA receptors or to the inventive GABA receptor complexes, to a metho d for qualitatively or quantitatively detecting the inventive nucleic acid sequences or the inventive proteins, and to a method for locating substances which specifically bind to an inventive GABA receptor or to an inventive nucleic acid sequence. The invention also relates to the use of the nucleic acid sequences and proteins. Finally, the invention relates to substances wi th which the activity of metabotropic GABA receptors can be influenced by influencing the stability of the protein heteromer.

Description

METABOTROPIC GABA RECEPTOR COMPLEX ISSUED FROM
THE CENTRAL NERVOUS SYSTEM
The present invention relates to isolated proteins which encode novel metabotropic GAGA receptors and which form a novel metabotropic GABA receptor complex (= protein heteromer) with GABA$ receptor proteins, and to nucleic acid sequences or recombinant nucleic acid constructs which encode the proteins.
The invention also relates to host organisms or transgenic animals which contain the nucleic acid sequences or the recombinant nucleic acid constructs and also to monoclonal or polyclonal antibodies which are directed against the isolated proteins.
The invention furthermore relates to a process for discovering substances which possess a specific binding affinity for the novel GABA receptors according to the invention or the GABA
receptor complexes according to the invention, to a process for qualitatively or quantitatively detecting the nucleic acid sequences according to the invention or the proteins according to the invention, and to a process for discovering substances which bind specifically to a GABA receptor according to'the invention or to a nucleic acid sequence according to the invention. The invention additionally relates to the use of the nucleic acid sequences and proteins.
y-Aminobutyric acid (GABA) is the inhibitory neurotransmitter which is primarily used in the vertebrate central nervous system.
GABA interacts with two types of receptor, i.e. GABAA and GABAB.
The effect of GABA on ionotropic receptors, i.e. the GABAA
receptors, is well characterized (Barnard et al., Trends Neurosci., 10, 1987: 502 - 509). These heteromeric complexes form anion channels which are opened in response to ligand binding.
The release of GABA leads, by way of activating these channels, to an inflow of chloride ions, i.e, an inhibitory post-synaptic flow, into the cell. GABAA receptors are the points of attack for a number of drugs such as benzodiazepines, barbiturates and more besides.(North (ed.), 1994, Ligand- and Voltage Gated Ion Channels, in: Handbook of receptors and channels, Vol. 2, CRC
Press, Inc. and Smith and Olson, Trends Neurosci., 16, 1995:
162 - 168].
Binding sites for GABA which exist independently of binding sites on GABAA receptors were demonstrated for the first time in 1981 (Hill and Bowery, Nature, 290, 1981: 149 - 152). They are located ~' 2 on GAGA receptors which are coupled intracellularly to G
proteins. By way of these G proteins, these receptors are coupled to neuronal potassium and calcium channels. These new GABA
receptors (= GABAH receptors) are also termed metabotropic GABA
receptors. These GABAB receptors are distributed through the central and peripheral nervous system (Ong et al., Life Sciences, Vol. 46, 1990: 1489 - 1501, Bowery et al., Drug Res., 42 (1), 1992: 215 - 223). These receptors are found both presynaptically and post-synaptically. At the presynapse, the metabotropic GABA
receptors (= GABAB receptors) control the release of different neurotransmitters such as GABA, L glutamate, noradrenaline, dopamine, serotonin, substance P, cholecystokinin, somatostatin and others. Ligands, agonists or antagonists of GABAH receptors which regulate the release of a specific neurotransmitter or neuropeptide can be used for redressing imbalances between different neurotransmitter systems, as occur in association with neurodegenerative diseases, excitotoxic symptoms accompanying neurological diseases and psychiatric diseases. GABAg receptors which activate various potassium channels by way of Gi proteins are found at the post-synapse. In transgenic mice, null mutations for such a channel lead to the loss of the late inhibition brought about by GABA and as a result to spontaneous convulsions.
GABAB receptors are involved in changes of synaptic efficiency which underlie learning and memory processes. GABA$ receptor agonists exhibit a positive effect in animal models for chronic pain and cocaine dependency. Antagonists have a positive effect in models of "absence epilepsy" (Settler et al., Curr. Opin.
Neurolbiol., 8, 1998: 345 - 350). Activation of GABA$ receptors dampens overstimulated neuronal connections. GABAB receptors are therefore suitable molecular targets for the treatment of epilepsy, stroke, dystonia, cognitive losses, acute and chronic pain, spongiform encephalitis, diseases of or injuries to the spinal cord, spasticity and other neurological diseases and also for the treatment of psychological disorders such as anxiety, depressive disorders, schizophrenia, migraine and others. They are also suitable for use as targets for the therapy of cocaine-or alcohol-dependent patients and as a point of attack for novel cognitive enhancers. Baclofen (lioresal), which is a GABAB
receptor agonist, is employed clinically for treating multiple sclerosis, spasticity and in association with the consequences of spinal cord injuries (Bowery, Annu. Rev. Pharmacol. Toxicol., 33, 1993: 109 - 147).
GABAB receptor agonists are also presumed to have positive effects in the peripheral nervous system (PNS), for example in association with inflammations and disorders of the respiratory tract. Substances which act on the GABA$ receptor are particularly v ' 3 suitable for the treatment of asthma, anaphylactic bronchospasm, airway hyperreactivity and pathological coughing. These substances can also have beneficial effects an functional disturbances or degenerative disorders of the retina such as, for example, retinitis pigmentosa. Such substances may also play an important part in the treatment of disorders of the gastrointestinal tract, in particular reflux esophagitis, gastric carcinogenesis, gastric ulcers and disorders characterized by irregular secretion of gastric acid or pepsinogen, but also for diabetes mellitus and other disorders of the pancreas and for disorders of the immune system.
Individual biochemical and pharmacological findings may possibly be interpreted as indicating that several GABAB receptor subtypes, possessing different functions, exist in the central nervous system. So far, the cDNA has been found for a GABAB receptor which occurs in two aminoterminal splice forms which hardly differ pharmacologically (Kaupmann et al., Nature, 386, 239-246, 1997, W097/46675). Following expression in heterologous systems, this GABA$ receptor exhibits similarly high affinities for the known GABA$ receptor antagonists as are found in the brain. By contrast, the affinities of GABA$ receptor agonists for this cloned GABAB
receptor fall below the values measured in the brain by about a factor of 100 (Kaupmann et al., Nature, 386, 239-246, 1997).
Froestl et al., J. Med. Chem. Vol. 38, 1995: 3297 - 3312 and 3313 - 3331 provide information on various GABAB receptor antagonists and agonists and their effect.
Since GABA$ receptors play a central role in various pathological processes of the central and peripheral nervous system, or are involved in such processes, they are sought-after targets for developing novel drugs.
It was therefore an object of the present invention to identify and characterize novel GABAB receptors or proteins which interact with the GABA$ receptors which possess binding sites which have as high an affinity as possible for GABAB receptor agonists and antagonists and which thereby make it possible to develop molecular test systems which can be used for screening, in a short period of time, many thousand different compounds for high-affinity substances. These substances, which have been characterized in this way and which interact specifically with the GABA$ receptor, are potential candidates for active compounds against diseases such as epilepsy, stroke, psychological disorders such as anxiety, manic-depressive disorders, schizophrenia, migraine and more besides.

:. . 4 We have found that this object is achieved with the protein heteromer according to the invention, which contains at least one GABA$ receptor protein and at least one protein having the amino acid sequence depicted in SEQ ID N0: 2 or SEQ ID NO: 4, or a sequence which can be obtained from this sequence by the substitution, inversion, insertion or deletion of one or more amino acid residues, with at least one of the essential biological properties of the protein depicted in SEQ ID NO: 2 or SEQ ID N0: 4, or of the protein heteromer, still being conserved, or with the isolated protein which contains the amino acid sequence depicted in SEQ ID N0: 2 or SEQ ID N0: 4, or a sequence which can be obtained from this sequence by the substitution, inversion, insertion or deletion of one or more amino acid residues, with at least one of the essential biological properties of the protein depicted in SEQ ID NO: 2 or SEQ ID
NO: 4 still being conserved.
The protein heteromers according to the invention are to be understood as being GABA receptor complexes, advantageously metabotropic GAGA receptor complexes, which contain at least one GABAB receptor protein and at least one protein having the amino acid sequence depicted in SEQ ID NO: 2 or SEQ ID NO: 4. Suitable GABA$ receptor proteins which can advantageously be present in the protein heteromers are described in W097/46675. In W097/46675, these GABA$ receptor proteins have the sequence designations or clone names "SEQ ID NO:1" or "GABABRla rat" (cloned from Rattus norvegicus), "SEQ ID N0:3" or "GABABRla/b human" (cloned from Homo Sapiens), "SEQ ID N0:5" or "GABABRlb rat" (cloned from Rattus norvegicus) and "SEQ ID N0:7" or "GABABRlb human" (cloned from Homo sapiens). These receptors and the document w097/46675 are hereby expressly incorporated by reference at this point.
The isolated proteins according to the invention are to be understood as being proteins which contain an amino acid sequence depicted in SEQ ID N0: 2 or SEQ ID NO: 4 or a sequence which can be obtained from this sequence by the substitution, inversion, insertion or deletion of one or more amino acid residues, with at least one of the essential biological properties of the protein depicted in SEQ ID N0: 2 or SEQ ID N0: 4 still being conserved.
In this connection, particular amino acids can, for example, be replaced with other amino acids having similar physicochemical properties (space-filling properties, basicity, hydrophobicity, etc.). For example, arginine residues are replaced with lysine residues, valine residues are replaced with isoleucine residues or aspartic acid residues are replaced with glutamic acid residues. However, one or more amino acids can also have their order reversed, be added or removed, or several of these _ procedures can be combined with each other. The proteins which are altered in this way as compared with SEQ ID NO: 2 or SEQ ID
NO: 4 possess at least 60%, preferably at least 70% and particularly preferably at least 90% sequence identity with the 5 sequences SEQ ID N0: 2 or SEQ ID N0: 4, as calculated using the "Altschul et al., J. Mol. Biol., 215, 403-410, 1990" algorithm.
The essential biological properties of the proteins or protein heteromers according to the invention are to be understood as being the transmembrane region(s), the aminoterminal region and, essentially, the carboxyterminal region of the protein alone or in the protein heteromer (see Figure 2). These protein regions enable the proteins or protein heteromers to have their special biological effect. These essential biological properties additionally comprise the high-affinity binding (Kd<lOnM) of specific synthetic or natural agonists and antagonists to the proteins according to the invention having the amino acid sequence depicted in SEQ ID NO: 2 or SEQ ID NO: 4, signal transmission to an intracellular G protein and interaction with the abovementioned, known GABAe receptors.
The proteins SEQ ID NO: 2 and 4 according to the invention are negatively coupled to the enzyme adenylate cyclase and therefore bring about, after activation by GABA or baclofen, a reduction in the forskolin-stimulated cAMP.production. This result proves that both proteins are functional GABA receptors. The decrease in cAMP
production mediated by the proteins according to the invention is pertussis toxin-sensitive, which suggests involvement of the Gi/
Go class of G proteins.
An important physiological property of native GABA$ receptors is activation of outwardly directed potassium currents by GIRKs (G-protein coupled inwardly recifying potassium channels). On recon-stitution of the proteins of SEQ ID N0: 2 or 4 according to the invention with GIRK1 and GIRR2 in HEK293 cells there is no mea-surable increase in the GIRK currents after activation with GABA
or baclofen. The protein heteromers according to the invention do, however, mediate after activation by GABA or baclofen a dis-tinct increase in the potassium conductivity through GIRK chan-nels in a pertussis toxin-sensitive manner.
The protein heteromers according to the invention are therefore essential constituents of the signal cascade which is induced by GAGA and leads to activation of GIRKs. The coupling of the GABA$
receptors with GIRKs therefore depends on the physical interac-tion in the protein heteromer according to the invention.

_ The domains which are responsible for the interaction in the pro-tein heteromer have been analyzed through deletion constructs and subsequent analysis in the two-hybrid system and in the GST pull-down assay. The interaction is mediated by two short domains which occur centrally in the intracellular c terminus of the re-spective protein. These domains relate to sequence regions of 35 and 32 amino acids respectively (amino acids 887-921 in the GABAB
receptor lA or amino acids 785-816 in SEQ ID N0: 2 and amino acids 786-817 in SEQ ID NO: 4) and have alpha-helical structures (based on secondary structure predictions [Garnier et al., J.
Mol. Biol., 120, 1978: 97-120j). The interaction between these domains mediates specifically for the formation of the heteromer, but not the formation of possibly homodimers or homomultimers.
The isolated protein, and its functional variants, can advantageously be isolated from the brains of mammals such as Homo sapiens or Rattus norvegicus. Homologs from other mammals are also to be understood as being functional variants.
The invention furthermore relates to nucleic acid sequences which encode the above-described proteins, in particular those proteins which have the primary structure depicted in SEQ ID NO: 2 or SEQ
ID N0: 4. The nucleic acid sequences from Rattus norvegicus and Homo sapiens are depicted in SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
The nucleotide sequences SEQ ID NO: 1 and SEQ ID N0: 3 according to the invention, or their functional equivalents such as allele variants, can be obtained following isolation and sequencing.
Allele variants are to be understood as being variants of SEQ ID
NO: 1 or SEQ ID NO: 3 which exhibit from 60 to 100 homology at the amino acid level, preferably from 70 to 100% homology, very particularly preferably from 90 to 1008 homology. Allele variants comprise, in particular, those functional variants which can be obtained by the deletion, insertion or substitution of nucleotides from the sequence depicted in SEQ ID NO: 1 or SEQ ID
NO: 3, with at least one of the essential biological properties still being conserved. Homologous or sequence-related nucleic acid sequences can be isolated from all the mammalian species, including man, using current methods, by means of screening for homology by hybridizing with a sample of the nucleic acid sequences according to the invention or parts thereof.
Functional equivalents are also to be understood as being homologs of SEQ ID NO: 1 or SEQ ID NO: 3, for example their homologs from other mammals, truncated sequences or single-stranded DNA or RNA of the coding and non-coding DNA
sequences.
Such functional equivalents can be isolated from other vertebrates, such as mammals, using the DNA sequences described in SEQ No: 1 or SEQ ID N0: 3, or parts of these sequences, and, for example, employing customary hybridization methods or the PCR
technique. These DNA sequences hybridize with the sequences according to the invention under standard conditions. For the hybridization, use is advantageously made of short oligonucleotides from the conserved regions, for example from the transmembrane regions or from the aminoterminal region, which oligonucleotides can be identified by making comparisons with other transmembrane proteins, especially other GAGA receptors, in a manner known to the skilled person. However, longer fragments of the nucleic acids according to the invention, or the complete sequences, can also be used for the hybridization. These standard conditions vary depending on the nucleic acid, oligonucleotide, longer fragment or complete sequence employed or depending on which type of nucleic acid, i.e. DNA or RNA, is used for the hybridization. Thus, the melting temperatures for DNA: DNA hybrids are, for example approx. 10°C lower than those for DNA: RNA
hybrids of the same length.
Standard conditions are to be understood, for example, as being, depending on the nucleic acid, temperatures of from 42 to 58°C in an aqueous buffer solution having a concentraticn of from 0.1 to 5 x SSC (1 x SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50% formamide, for example 42°C in 5 x SSC, 50% formamide. Advantageously, the hybridization conditions for DNA:DNA hybrids are 0.1 x SSC and temperatures of from about 20°C to 45°C, preferably from about 30°C to 45°C. For DNA: RNA hybrids, the hybridization conditions are advantageously 0.1 x SSC and temperatures of from about 30°C to 55°C, preferably of from about 45°C to 55°C. These temperatures which are indicated for the hybridization are melting temperature values which are calculated, by way of example, for a nucleic acid having a length of approx. 100 nucleotides and a G + C content of 50%, in the absence of formamide. The experimental conditions for DNA
hybridization are described in specialist genetics textbooks such as Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated using formulae known to the skilled person, for example depending on the length of the nucleic acids, the nature of the hybrids or the G + C content.
The skilled person can obtain further information regarding hybridization from the following textbooks: Ausubel et al. (eds), 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids ... 8 Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed.), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.
Homologs of the sequences SEQ ID N0: 1 and SEQ ID N0: 3 are also to be understood as being derivatives such as promoter variants.
The promoters, which are jointly or individually located upstream of the given nucleotide sequences, can be altered by one or more nucleotide replacements, by (an) insertions) and/or (a) deletions) without, however, the functionality or activity of the promoters being impaired. Furthermore, the activities of the promoters can be increased by altering their sequences, or the promoters can be completely replaced with more active promoters, including those from organisms of another species.
Derivatives are also advantageously to be understood as being variants whose nucleotide sequences have been altered in the -1 to -1000 region upstream of the start codon such that expression of the gene and/or expression of the protein is altered, preferably increased. Furthermore, derivatives are also to be understood as being variants which have been altered at their 3' end.
In order to be able to achieve optimum expression of heterologous genes in organisms, it is advantageous to alter the nucleic acid sequences in accordance with the specific codon usage employed in the organism. The codon usage can easily be determined with the aid of computer analyses of other known genes from the organism concerned.
It is furthermore advantageous to functionally link the nucleic acid SEQ ID NO: 1 or SEQ ID NO: 3 according to the invention on their own, or the nucleic acid SEQ ID NO: 1 or SEQ ID NO: 3 and a sequence which encodes a GABA$ receptor protein, to at least one genetic regulatory element to form the recombinant nucleic acid constructs according to the invention.
For this, the nucleic acid sequences according, to the invention are usually functionally linked to genetic regulatory elements such as transcription and translation signals. Depending on the desired application, this linking can lead to an increase or a reduction in gene expression. The recombinant nucleic acid constructs which have been prepared in this way are then used for transforming host organisms. In addition to these new regulatory sequences, the natural regulation of these sequences can still be present upstream of the actual structural genes and, where appropriate, have been altered genetically such that the natural regulation has been switched off and expression of the genes has been increased. However, the gene construct can also be assembled in a simpler manner, meaning that no additional regulatory signals are inserted upstream of the sequences and that the natural promoter, together with its regulation, is not removed.
Instead of this, the natural regulatory sequence is mutated such that there is no longer any regulation and gene expression is increased. Additional, advantageous regulatory elements can also be inserted at the 3' end of the nucleic acid sequences. The nucleic acid sequences for the sequences SEQ ID N0: 1 or SEQ ID
NO: 3 and/or for the GABAB receptor proteins can be present in the gene construct in one or more copies, or can be located on separate gene constructs.
Regulatory sequences which are advantageous for the process according to the invention are present, for example, in promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, ~,-PR or 7~-PL promoters, which are advantageously used in Gram-negative bacteria. Other advantageous regulatory sequences are present, for example, in the Gram-positive promoters such as amy and SP02, in the yeast promoters such as ADC1, MFa, AC, P 60, CYC1 and GAPDH, or in mammalian promoters such as CaM-kinaseII, CMV, nestin, L7, BDNF, NF, MBP, NSE, ~-globin, GFAP, GAP43, tyrosine hydroxylase, kainate receptor subunit 1 and glutamate receptor subunit B.
In principle, all natural promoters, together with their regulatory sequences, can be used like those mentioned above. In addition to this, use can also advantageously be made of synthetic promoters.
These regulatory sequences are intended to facilitate deliberate expression of the nucleic acid sequences and protein expression.
Depending on the host organism, this can, for example, mean that the gene is only expressed or overexpressed after induction or that it is expressed and/or overexpressed immediately.
In this context, the regulatory sequences or factors can preferably exert a positive influence on expression and thereby increase it. Thus, the regulatory elements can advantageously be reinforced at the transcriptional level by using strong transcription signals such as promoters and/or enhancers. In addition to this, however, it is also possible to reinforce translation by, for example, improving the stability of the mRNA.

Enhancers are to be understood, for example, as being DNA
sequences which bring about increased expression by improving the interaction between the RNA polymerase and the DNA. Additional regulatory sequences which may be mentioned by way of example are 5 locus control-regions and silencers, or particular part sequences thereof. These sequences may advantageously be used for tissue-specific expression.
A preferred embodiment is the linking of the nucleic acid 10 sequence according to the invention to a promoter, with the promoter coming to lie 5' upstream. Other regulatory signals, such as 3' located terminators or polyadenylation signals or enhancers, can be used functionally in the nucleic acid construct.
The term "recombinant nucleic acid construct or gene construct"
according to the invention is also to be understood as meaning complete vector constructs. These vector constructs or vectors are used for expression in a suitable host organism.
Advantageously, the nucleic acids according to the invention and/or the genes for the GABAB receptors are inserted into a host-specific vector which enables the genes to be expressed optimally in the chosen host. Vectors are well known to the skilled person and can be identified, for example, from the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Vectors are also to be understood as being, apart from plasmids, all the other vectors which are known to the skilled person, such as phages, viruses such as SV40, CMV, baculoviras and adenovirus, transposons, IS elements, phasmids, phagemids, cosmids and linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally. Linear DNA is advantageously used for integration in mammals.
The expression of the nucleic acid sequences according to the invention or of the recombinant nucleic acid construct can advantageously be increased by increasing the gene copy number and/or by reinforcing regulatory factors which exert a positive influence on gene expression. Thus, regulatory elements can preferably be reinforced at the transcriptional level by using stronger transcription signals such as promoters and enhancers.
However, in addition to this, it is also possible to reinforce translation by, for example, improving the stability of the mRNA
or increasing the efficiency with which this mRNA is read off on ribosomes.

~091~00001 CA 02343574 2001-03-13 In order to increase the gene copy number, the nucleic acid sequences, or homologous genes, can, for example, be incorporated into a nucleic acid fragment or into a vector which preferably contains the regulatory gene sequences, or analogously acting promoter activity, which is/are assigned to the respective genes.
Use is, in particular, made of those regulatory sequences which reinforce gene expression.
The nucleic acid sequences according to the invention can be cloned, together with the sequences encoding the GABAB receptors, into a single vector and subsequently expressed in the desired organism. Alternatively, each of the described nucleic acid sequences and the sequences encoding the GABAB receptors can also in each case be introduced into an individual vector and these vectors can then be introduced separately into the respective organism using customary methods such as transformation, transfection, transduction, electroporation or a particle gun.
In addition to this, the nucleic acid construct according to the invention or the nucleic acids according to the invention can also be expressed in the form of therapeutically or diagnostically suitable fragments. In order to generate the recombinant protein, use can be made of vector systems or oligonucleotides which extend the nucleic acids or the nucleic acid construct by adding on defined nucleotide sequences and thereby encode modified polypeptides which are used to simplify purification. Hexahistidine anchors, or epitopes which can be recognized as the antigens for different antibodies, are, for example, known in the literature as being tags of this nature (Studier et al., Meth. Enzymol., 185, 1990: 60 - 89 and Ausubel et al. [eds.] 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York).
In principle, all organisms which enable the nucleic acids according to the invention, their allele variants, their functional equivalents or derivatives, or the recombinant nucleic acid construct on its own or together with a sequence which encodes GABAB receptor proteins, to be expressed are suitable for use as host organisms. Host organisms are, for example, to be understood as being bacteria, fungi, yeasts or plant or animal cells. Preferred organisms are bacteria, such as Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms, such as Saccharomyces cerevisiae or Aspergillus, and higher eukaryotic cells from humans or animals, for example COS, Hela, HEK293, Sf9 or CHO cells.

If desired, the gene product can also be expressed in transgenic organisms such as transgenic animals, e.g. mice, rats, sheep, cattle or pigs. Transgenic plants are also conceivable in principle. The transgenic organisms can also be so-called knockout animals.
In this context, the transgenic animals can contain a functional or non-functional nucleic acid sequence according to the invention or a functional or non-functional nucleic acid construct on its own or in combination with a functional or non-functional sequence which encodes GABAB receptor proteins.
Another form, according to the invention, of the above-described transgenic animals is constituted by transgenic animals in whose germ cells, or the entirety or a part of the somatic cells, or in whose germ cells and the entirety or a part of the somatic cells, the nucleotide sequence according to the invention has been altered by recombinant methods or interrupted by inserting DNA
elements.
The combination of the host organism and the vectors, such as plasmids, viruses or phages, which are appropriate for the organism, such as, for example, plasmids containing the RNA
polymerase/promoter system, the phages 1, Mu, or other temperate phages, or transposons, and/or other advantageous regulatory sequences, forms an expression system. The term expression systems is preferably to be understood as meaning, for example, the combination of mammalian cells such as CHO cells or HEK293 cells and vectors which are suitable for mam.~nalian cells, such as pcDNA3neovector and CMV vector.
In-situ hybridization with the sequence SEQ ID NO: 2 or parts thereof, gave strong expression in the hippocampus, the cortex and the cerebellum and also in the thalamic nuclei (,see Figure 1 and examples). Figures la and lb depict the analysis of~ the expression of the mRNA corresponding to SEQ ID N0: 1. la shows the Northern blot, while lb shows the in-situ hybridization (see Examples 3 and 4). The pattern of expression overlaps with that of the GABAH receptor and indicates that the protein depicted in SEQ ID N0: 2 or SEQ ID N0:4 has an important central nervous function. The hippocampus is the crucial brain structure for storing new memory contents. A protein having the sequence SEQ ID
N0: 2 is consequently an interesting target for understanding in relation to learning and memory and for developing new cognitive enhancers. As part of the limbic system, the hippocampus also exerts an influence on moods and feelings. Drugs directed against SEQ ID NO: 2 or SEQ ID N0:4 and their functional equivalents, - _ homologs or derivatives consequently constitute potential antidepressants or anxiolytics and can be used in association with cognitive disorders. Finally, the hippocampus is deeply involved in temporal lobe epilepsies, thereby making a protein possessing the sequence SEQ ID N0:2 or SEQ ID N0:4 an attractive target for novel drugs against this frequently occurring disorder. The cortex contains regions which integrate and process sensory information and convert it into suitable reactions. These sensory and motor centers are also often the starting points for epileptic fits. Exerting a deliberate influence on the proteins of the invention or the protein heteromer of the invention might lower the probability of convulsions in epilepsy patients. The thalamic nuclei are connected in series upstream of the cortex, and integrate the perceptions which are received by the sense organs and pass them on to cortical structures. They are frequently the starting point for generalized seizures. The fact that the proteins according to the invention or the protein heteromer is/are strongly expressed in the thalamic nuclei indicates that its/their activation or inhibition may contribute to alleviating seizures in epilepsy patients. The cerebellar connections are substantially responsible for the fine coordination of movements. Ataxias and other motor disorders might be due to the deregulation of a protein having the nucleic acid sequence according to the invention. The protein heteromers or proteins according to the invention consequently represent interesting targets for developing novel substances which can be used for producing medicaments for treating diseases such as neurological disorders, such as epilepsy, stroke, psychological disorders, such as anxiety, manic-depressive disorders, migraine, cognitive losses and other neurological disorders.
Examples of substances which exhibit an effect vis-a-vis GABAB
receptors are baclofen and its derivatives, as agonists, and phaclofen and saclofen and derivatives, as antagonists. These substances, and other active substances, are to be found in J.
Med. Chem., 38, 1995: 3313 - 3331, J. Med. Chem., 38, 1995:
3297 - 3312 and w097/46675. These substances very probably also have an activity vis-a-vis the protein heteromer according to the invention or the protein according to the invention. Presumably, the proteins according to the invention (= protein heteromer +
isolated protein) can be used to facilitate the development of substances having a more powerful agonistic effect. The novel proteins can be used to develop substances which are more selective.
The gene for the already known GABAe receptor is located in the vicinity of the chromosomal locus which is associated with - juvenile, myoclonic epilepsy. This correlation might also make it possible to develop a novel method for diagnosing this widespread form of epilepsy. The same applies to the proteins according to the invention. The nucleotide sequences SEQ ID N0: 1 and SEQ ID
N0: 3 can be used to isolate mRNA genes encoding these nucleic acids or their functional equivalents, homologs or derivatives in the murine and human genomes, by means of homology screening using customary methods, and then to map these genes and correlate them with markers for human hereditary diseases. This then makes it possible to identify the gene which is the cause of particular hereditary diseases, thereby considerably simplifying the diagnosis of these diseases and making it possible to develop new therapeutic approaches. It is thus possible to diagnose hereditary diseases using the nucleic acids as markers.
The invention also relates to the use of the nucleic acids according to the invention, or parts thereof, for gene therapy.
Sequences which are complementary to the nucleic acids according to the invention, or parts thereof, can also be used for gene therapy.
A further possibility for using the nucleotide sequence, or parts thereof, is that of producing transgenic or knockout, or conditional or region-specific knockout animals, or specific mutations in recombinantly modified animals (Ausubel et al.
[eds). 1998, Current Protocols in Molecular Biology, John Wiley &
Sons, New York and Torres et al., [eds.), 1997, Laboratory protocols for conditional gene targeting, Oxford University Press, Oxford). Animal models, which provide further valuable information about the (patho)physiology of the sequences according to the invention, either on their own or complexed with the GABAe receptor, can be generated by way of transgene overexpression or genetic mutation (null mutation or specific deletions, insertions or modifications) elicited by homologous recombination in embryonic stem cells. Animal models which have been produced in this way can represent essential test systems for evaluating novel therapeutic agents which exert an effect on signal transduction by GABAB receptors.
The interaction of the known GABA$ receptor with the novel 7 transmembrane region protein according to the invention, which has been described and which was discovered using the two-hybrid system, is of great physiological importance. This surprising finding opens up new exceptional opportunities for treatment with regard to the abovementioned neurological and psychological disorders which are connected with the GABAH receptor. Low molecular weight effectors or peptides which exert a positive or negative influence on this interaction are active compounds which intervene in GABAergic signal transduction and can therefore be used as a new class of drugs. There has been no previous knowledge of a direct molecular interaction between two different metabotropic receptors. There has likewise been no previous description of substances which exert an influence on the interaction between two different metabotropic receptors and thereby modulate signal transduction via these receptors. The protein heteromer according to the invention can therefore be used to develop novel active compounds and active compound classes.
The nucleic acid sequence according to the invention, the nucleic acid construct, a protein heteromer according to the invention, or the protein, can be used to identify proteins which exhibit specific binding affinities for the protein heteromer or for the protein, or to identify nucleic acids which encode proteins which exhibit specific binding affinities for the protein heteromer or the protein. The two-hybrid system or other biochemical methods, either on their own or in combination, are advantageously used for this purpose. In this way, it is possible to determine interaction domains of metabotropic receptors and, as a consequence, to determine points for pharmacotherapeutic intervention.
The invention therefore relates to the use of the two-hybrid system or biochemical methods for identifying the interaction domains of metabotropic receptors and to the use for pharmacotherapeutic intervention.
Structural analyses of the protein heteromer or the protein according to the invention can be used in a deliberate manner to find substances which exhibit a specific binding affinity.
The described sequences SEQ ID NO: 1 and SEQ ID NO: 3 make it possible, with the aid of the two-hybrid system or other assays, to circumscribe amino acids which are responsible for the interaction and to find substances which can be used for exerting an influence on the interaction between the two metabotropic receptors. Irrespective of the special case which is described here with regard to the GABAB receptor, the use of substances which influence the physical interaction of two metabotropic receptors for treating diseases represents a novel pharmacological principle.

The invention further relates to substances, in particular GABAB
receptor antagonists, which specifically reduce or prevent the natural interaction of the GABAB protein with the protein of SEQ
ID NO: 2 or 4.
Such substances preferentially bind to the following sequence re-gions:
(i) to amino acid sequence 887-921 of the GABA receptor lA or (ii) to amino acid sequence 785-816 of SEQ ID N0: 2 or (iii) to amino acid sequence 786-817 of SEQ ID NO: 4.
Besides substances which bind to these sequences, these polypep-tides themselves and parts of these polypeptides are also suit-able as substances interfering with or preventing the interac-tion, in particular polypeptides which have a sequence of at least 5 amino acids of one of these sequences (i), (ii) and (iii).
The invention further relates to a process for discovering substances having a specific binding affinity for the protein heteromer or protein according to the invention, which process comprises the following steps:
a) incubating the proteins) with the substance to be tested, and b) detecting the binding of the substance to be tested to the protein.
The binding is detected by measuring the antagonization or agonization of the GABAB receptor activity or by measuring a physiological effect, such as a change in the concentration of calcium, cAMP or IP3, or in the membrane potential.
Other embodiments of the invention are a process for discovering substances which inhibit or reinforce the interaction of proteins having amino acid sequences as depicted in SEQ ID N0: 2 or SEQ ID
NO: 4 with other metabotropic receptors; a process for discovering substances which inhibit or reinforce the interaction of ligands with the protein heteromer according to the invention or the proteins according to the invention having amino acid sequences such as SEQ ID NO: 2 or SEQ ID N0: 4, or a process for discovering substances which inhibit or reinforce the interaction of proteins having amino acid sequences such as SEQ ID NO: 2 or ~ 17 SEQ ID NO: 4 with G proteins or other signal transduction molecules. The interaction of proteins having the amino acids according to the invention can be detected using the two-hybrid system. Furthermore, the processes can be carried out by expressing the proteins in eukaryotic cells and linking to a reporter assay for the activation of the GABAB receptor. Change in the cAMP level or in the membrane potential is, for example, detected in this context.
The invention furthermore relates to a process which uses specific agonists or antagonists for qualitatively and quantitatively determining proteins having amino acid sequences such as SEQ ID N0: 2 or SEQ ID N0: 4. GABAB receptor-ligand binding is used for the detection in this case.
The protein activity of the proteins having the sequences SEQ ID
NO: 2 or SEQ ID N0: 4 can be determined using antibodies. For this reason, the invention additionally relates to a process for quantifying the protein activity of a protein having the sequences SEQ ID NO: 2 or SEQ ID N0: 4.
The regulatory sequences of the nucleic acids according to the invention, in particular the promoter, the enhancers, locus control regions and silencers, or respective part sequences thereof, can be used for the tissue-specific expression of this and other genes. This provides the possibility of carrying out the brain-specific gene expression of nucleic acid constructs.
In order to isolate a DNA fragment which contains the regions which regulate the transcription of the sequences SEQ ID N0:1 or SEQ ID N0: 3, a genomic library is first of all screened with a cDNA probe which is located as far as possible in the 5' direction. A homology search, with which the skilled person is familiar, is carried out for this purpose (Ausubel et al. [eds.], 1998, Current Protocols in Molecular Biology, John wiley & Sons, New York). The transcription start is then identified on the isolated DNA fragment. The region upstream of the transcription start is then linked to a reporter gene such as ~-galactosidase or GFP (= green fluorescent protein) and tested in cells or in transgenic animals such as mice to see whether it gives rise to the expression pattern which is specific for SEQ ID NO:1 or SEQ
ID NO: 3 (Ausubel et al., see above). The reporter gene can then be linked to other cDNAs in order to construct animal models in which the respective cDNA is expressed in a region-specific manner (see, for example, Oberdick et al., Science, 248, 1990:
223 - 226).

~ 18 The amino acid sequences SEQ ID N0: 2 or SEQ ID N0: 4 can be used to generate synthetic peptides which are employed as antigens for producing antibodies. It is also possible to use the polypeptide, or fragments thereof, for generating antibodies. Antibodies are understood to mean polyclonal, monoclonal, human or humanized or recombinant antibodies, or fragments thereof, single-chain antibodies or synthetic antibodies. In principle, antibodies according to the invention, or their fragments, are to be understood as being all immunoglobulin classes, such as IgM, IgG, IgD, IgE or IgA, or their subclasses, such as the subclasses of IgG, or mixtures thereof. IgG and its subclasses, such as IgGl, IgG2, IgG2a, IgG2b, IgG3 and IgGM, are preferred. The IgG subtypes IgGl/x or IgG2b/x are particularly preferred. Fragments which may be mentioned are all truncated or modified antibody fragments which possess one or two binding sites which are complementary to the antigen, such as antibody moieties having a binding site which corresponds to that of the antibody and which is formed from a light chain and a heavy chain, such as Fv, Fab or F(ab')Z
fragments, or single-stranded fragments. Truncated double-stranded fragments such as Fv, Fab or F(ab')2, are preferred. These fragments can be obtained, for example, enzymically, by cleaving off the Fc moiety of the antibodies with enzymes such as papain or pepsin, by means of chemical oxidation, or by genetic manipulation of the antibody genes. Genetically manipulated untruncated fragments can also advantageously be used.
The antibodies or fragments may be used on their own or in mixtures.
The antibody genes for the genetic manipulations can be isolated in a manner known to the skilled person, for example from the hybridoma cells (Harlow, E. and Lane, D. 1988, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press, N.Y.; Ausubel et al., [eds], 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York). For this, antibody-producing cells are propagated and, when the cells have reached an adequate optical density, the mRNA is isolated from the cells in a known manner by lyzing the cells with guanidinium thiocyanate, acidifying with sodium acetate, extracting with phenol and chloroform/isoamyl alcohol, precipitating with isopropanol and washing with ethanol.
cDNA is then synthesized from the mRNA using reverse transcriptase. The synthesized cDNA can be inserted into suitable animal, fungal, bacterial or viral vectors, and expressed in the appropriate host organisms, either directly or following genetic manipulation, for example by means of site-directed mutagenesis, the introduction of insertions, inversions or deletions, or the substitution of bases. Preference is given to bacterial or yeast - _ vectors such as pBR322, pUCl8/19, pACYC184, lambda or yeast mu vectors, for cloning the genes, and to expression in bacteria, such as E. coli, or in yeast, such as Saccharomyces cerevisiae.
Specific antibodies against the proteins according to the invention can be suitable for use in association with neurological or psychiatric disease syndromes both as diagnostic reagents and as therapeutic agents.
Furthermore, the cDNA, the genomic DNA, the regulatory elements of the nucleic acid sequences according to the invention and the polypeptide, and also part fragments thereof, can be used, in recombinant or non-recombinant form, for elaborating a test system. This test system is suitable for measuring the activity of the promoter or of the protein in the presence of the test substance. In this context, these test systems are preferably simple measuring methods (colorimetric, luminometric, fluorescence-based or radioactive) which permit rapid measurement of a large number of test substances (Bohm, Klebe, Kubinyi, 1996, Wirkstoffdesign (Active Compound Design), Spektrum-Verlag, Heidelberg). The test systems described enable chemical libraries to be screened for substances which have agonistic or antagonistic effects on SEQ ID N0: 2 or SEQ ID N0: 4 or the novel GABAH receptor complex consisting of the already described GABA$
receptor and the protein described in SEQ ID NO: 2 or SEQ ID
NO: 4. The identification of such substances constitutes the first step on the route to identifying novel drugs which act specifically on GABAergic signal transduction.
An alternative route for developing active compounds which interact with the novel GABAB receptor consists in rational drug design (Bohm, Klebe, Kubinyi, 1996, Wirkstoffdesign (Active Compound Design), Spektrum-Verlag, Heidelberg). In this case, the structure, or a part structure, of the protein depicted in SEQ ID
NO: 2 or SEQ ID N0: 4, insofar as it is available, or a model of the structure which is constructed by computers, is used, with the support of molecular modeling programs, to find structures which can be predicted to have a high affinity for the GABA$
receptor. These substances are then synthesized and tested.
High-affinity, selective substances are tested for their use as drugs against epilepsy, stroke and other neurological disorders.
The determination of quantity, activity and distribution of the novel GABA$ receptor complex or of the protein depicted in SEQ ID
N0: 2 or SEQ ID NO: 4, or its underlying mRNA, in the human body can be used for diagnosis, determination of predisposition and monitoring in association with particular disorders. Similarly, the sequence of the cDNA for the sequences SEQ ID NO: 2.or SEQ ID
NO: 4, and of the genomic DNA, can be invoked for making predictions with regard to the genetic causes and predispositions for particular disorders. Both DNA/RNA probes and a very wide variety of antibodies can be used for this purpose. In this context, the described nucleotide sequence SEQ ID NO: 1 or SEQ ID
NO: 3, or parts thereof, is used, in the form of suitable probes, for revealing point mutations or deletions/insertions/
rearrangements.
The present nucleic acid sequence SEQ ID N0: 1 or SEQ ID NO: 3, its functional equivalents, homologs or derivatives, the protein which it encodes (SEQ ID N0: 2 or SEQ ID N0: 4), or the protein heteromer according to the invention, and also reagents derived therefrom (oligonucleotides, antibodies and peptides) can be employed for the diagnosis and therapy of neurological disorders.
It also becomes possible to diagnose and treat genetic predispositions for particular neurological disorders such as epilepsy, stroke, psychological disorders, such as anxiety, manic-depressive disorders, migraine, cognitive losses and other neurological disorders. Furthermore, it is possible to monitor the treatment of the abovementioned disorders.
The invention furthermore relates to a process for qualitatively and quantitatively detecting a nucleic acid according to the invention in a biological sample, which process comprises the following steps:
a) incubating a biological sample with a kr_own quantity of nucleic acid according to the invention or a known quantity of oligonucleotides which are suitable for use as primers for amplifying the nucleic acid according to the invention, b) detecting the nucleic acid according to the invention by specific hybridization or PCR amplification, c) comparing the quantity of hybridizing nucleic acid or of nucleic acid obtained by PCR amplification with a quantity standard.
In addition, the invention relates to a process for qualitatively and quantitatively detecting a protein heteromer according to the invention or a protein according to the invention in a biological sample, which process comprises the following steps:
a) incubating a biological sample with an antibody which is specifically directed against the protein heteromer or against the protein according to the invention, b) detecting the antibody/antigen complex, c) comparing~the quantities of the antibody/antigen complex with a quantity standard.
The standard is normally a biological sample which is withdrawn from a healthy organism.
The invention furthermore relates to a process for discovering substances which bind specifically to a protein having an amino acid sequence SEQ ID N0: 2 or SEQ ID NO: 4, which process comprises one or more of the following steps:
a) expressing the protein in eukaryotic or prokaryotic cells, b) incubating the protein with the substances to be tested, c) detecting the binding of a substance to the receptor or detecting an effect on the receptor function.
In addition, the invention relates to a process for discovering substances which bind specifically to a protein having an amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4, or to a nucleic acid sequence according to SEQ ID N0: 1 or SEQ ID N0: 3 and thereby elicit inhibitory or activating functional effects on GABAergic signal transmission in central nervous neurones.
Depending on the neurotransmitter system (e. g. GABA or glutamate) in which the GABAB receptor is involved, an increased or decreased GABAB receptor activity can lead to an imbalance between the neurotransmitter systems and often to a neuronal superexcitation which characterizes a large number of neurological disorders such as epilepsy, stroke and their sequelae, and others besides.
Deficient neuronal activity, which characterizes dementias, for example, can be the result when presynaptic GABAB receptors at glutaminergic synapses are hyperactivated and inhibit transmitter release so strongly that transmission of the stimulus across the synapse is no longer possible.
Several methods can be employed for effecting a replacement in situations where the activity of the protein according to the invention or of the GABAB receptor supplemented with SEQ ID NO: 2 or SEQ ID N0: 4 is in short supply. In the first place, the natural or recombinant protein can be administered directly or, by means of suitable procedures, in the form of its encoding nucleic acid (i.e. DNA or RNA). Both viral and non-viral vehicles can be used for this purpose. Another way is that of stimulating the endogenous gene by means of suitable substances. Such substances can be found, for example, by ascertaining their effect on the transcription elements of the novel GABA$ receptor gene.
In situations in which the activity of the GABAB receptor comprising a protein having the sequence SEQ ID NO: 2 or SEQ ID
NO: 4, or of a protein having SEQ ID NO: 2 or SEQ ID N0: 4 alone, is in excess, it is possible to employ specific, synthetic or natural, competitive and non-competitive antagonists against the protein having the sequence SEQ ID N0: 2 or SEQ ID NO: 4 or antibodies or antibody fragments against the protein having the sequence SEQ ID NO: .2 or SEQ ID N0: 4 or against the protein heteromer. Furthermore, both antisense molecules or ribozymes or oligonucleotides and low molecular weight compounds can be used to inhibit the GABAH receptor activity or the activity of the protein having the sequence SEQ ID N0: 2 or SEQ ID NO: 4.
Examples The nucleotide sequence having the structure depicted in SEQ ID
N0: 1 was identified from a cDNA library prepared from rat brain.
The nucleotide sequence was found while searching for proteins which interact with the intracellular carboxyterminus of the known GABAB receptor. In the experiment, a rat brain cDNA library was screened by the two-hybrid system for partners which interacted with the carboxyterminus of the above-described GABAB
receptor. Several overlapping fragments of an unknown cDNA were found. These fragments were used to isolate a 5 kb fragment of the unknown cDNA from a rat hippocampus cDNA library by means of homology screening and subsequently sequenced it. The cDNA
sequence which was obtained in this way contains the complete region encoding the sequence SEQ ID N0: 2. The discovery and molecular characterization of partners which interact with the known cloned GABAB receptor makes it possible to achieve a better understanding of the physiological properties and biochemical and pharmacological diversity of the GABA$ receptor, and also to obtain new specific points of attack for pharmacotherapeutic interventions.
Analysis of the sequence of the polypeptide encoded by the present cDNA (= SEQ ID N0: 1) indicates that this polypeptide is a metabotropic receptor. It contains an aminoterminal signal sequence [amino acids 1-40 (G. von Heijne, N.A.R., 14, 4683, 1986)] and seven characteristically arranged hydrophobic regions which very probably span the plasma membrane. The.presence of these seven hydrophobic regions is a characteristic of metabotropic receptors. Use of the BLAST program (BLASTP

2.Oa19-WashU version [05 Feb 1998]) to compare (Altschul et al., J. Mol. Biol., 215, 403-410, 1990) the amino acid sequence SEQ ID
NO: 2 with the sequences of the open reading frames of the translated public nucleotide data bases (nrdb) showed similarities to the above-described GABAH receptor (best score:
36$ sequence identity over 804 amino acids with GABAB receptor 1B
and 36~ sequence identity over 744 amino acids with GABAB receptor lA) and also a substantially lower similarity to the sequences of metabotropic glutamate receptors and calcium-sensitive receptors.
The similarity extends over the aminoterminal region and, in particular, over the transmembrane regions, but not over the carboxyterminal,.intracellular region (see..Figure 2). The protein described in SEQ ID NO: 2 is consequently a novel metabotropic receptor which either alone or in a complex with the known GABAH
receptor mediates signal transmission in the CNS, or regulates this transmission.
Figure 2 shows a comparison of the sequence of the protein according to the invention having the sequence SEQ ID N0: 2 with those of the known GABAB receptor proteins (= GBR) lA and 1B
(Kaupmann et al. 1997, see above). Identical amino acids are shown with a black background while conservative changes are shown by light-gray shading. The seven transmembrane regions (= TM1 to TM7) are marked by overlying lines.
The analysis of the distribution of the mRNA from which the cDNA
sequence SEQ ID NO: l originated was carried out on rat brain sections by means of Northern blotting and in-situ hybridization.
An analysis carried out in 10 different rat tissues indicated a brain-specific expression of an mRNA of 5-6 kb in size. A smaller mRNA can be detected on a very small scale in rat testis (see Figure lj. The in-situ hybridization indicated strong expression in the hippocampus, the cortex, the cerebellum and in the thalamic nuclei. The pattern of expression overlaps with that of the GABA$ receptor and points to the protein depicted in SEQ ID
NO: 2 having an important central nervous function.
Unless otherwise indicated, the experiments were carried out in accordance with the instructions given in "Ausubel et al.(eds.), 1998. Current Protocols in Molecular Biology. John Wiley & Sons, New York".

Example 1 Two-hybrid search using the GABA$ receptor 1 carboxy terminus The cDNA encoding the carboxy terminus of the GABAH receptor lA
(amino acids 857-960, accession no. Y10369, EMBL database) was amplified from rat brain cDNA in a polymerase chain reaction (PCR) using the specific primers GABA-CT5' (5'-GCGAATTCCGCAGGCTGATCACCCGAGGG-3') and GABA-CT3' (5'-GCAGTCGACTCACTTGTAAAGCAAATGTACTCG-3'), then restricted with the enzymes EcoRI and SalI and after that cloned, by way of the protruding ends, into a vector, pGBT (from Clontech), which had been previously cut with the enzymes EcoRI and SalI. The resulting DNA construct (pGBT-GABAH receptor 1) encodes a protein in which the Gal4-DNA-binding domain is fused to the C terminus of the GABAH receptor. The yeast strain HF7c (from Clontech) was transformed with this construct. The resulting yeast strain was transformed with a rat brain cDNA library (Kornau et al., Science 269, 1737-1740, 1995) in the vector pGAD (from Clontech), and 4x106 transformants were plated out on tryptophan/leucine/
histidine deficient medium. After 3, 4 and 5 days of growth at 30°C, colonies having a diameter of more than 2 mm were singled out and stained with XGal. A total of 7 colonies were found to be His3 and lacZ positive (pGAD-posl-7). The respective cDNA from each of these colonies was amplified from the pGAD vector using the vector-specific primer Ga14AD3' (5'-AAGAGATCCTAGAACTAGTGGATC-3') and T7 (5'-CGTAATACGACTCACTATAGGGCG-3'), and the amplicon was sequenced.
The sequence analysis yielded 5 different overlapping fragments (nucleotide sequences 2399-3102 (a), 2432-3102 (b), 2447-3102 (c), 2462-3102 (d), 2468-3102 (e) in the sequence SEQ ID NO: 1).
The pGAD plasmid DNA was purified from two different positive clones (a and e) and cotransformed into the yeast strain HF7c together with different pGBT constructs. It was only possible to observe activation of the reporter genes His3 and lacZ in combination with the construct pBGT-GABAB receptor 1.
Example 2 Cloning the cDNA for the novel GABAH receptor component SEQ ID
NO: 2 A cDNA fragment which was obtained from the two-hybrid search as described in Example 1 (a, nucleotides 2399-3102 in sequence SEQ
ID N0: 1) was radioactively labeled with a-32P-dCTP using a random-primed labeling kit (Boehringer Mannheim) in accordance _ with the manufacturer's instructions. The radioactive probe, which had been denatured by heating, was hybridized for 16 hours (42°C, SxSSC, 50% formamide) on 18 nitrocellulose filters onto each of which 40,000 plaques from a rat hippocampus cDNA library in bacteriophage ~, had been transferred; the filters were then washed several times, at 60°C, with 0.2xSSC. Out of 30 positive clones, 6 were singled out and their phage DNA was isolated and mapped. The two longest cDNA fragments (5 kb) were sequenced completely. They contain an open reading frame for the amino acid sequence SEQ ID NO: 2. Analysis of the sequences of these two lambda cDNA clones showed four differences in the coding sequence, 3 of which represent silent mutations (nucleotide 696, C to T, nucleotide 1104, T to C, nucleotide 2295, C to T), while the remaining difference encodes an additional proline in the aminoterminal signal sequence (insertion of CCG at nucleotide 171/172). Accordingly, the second clone has a sequence which is one amino acid (PRO) longer than SEQ ID NO: 2.
Example 3 Expressing the mRNA for the novel GABA$ receptor component in rat tissues A cDNA fragment which was obtained from the two-hybrid search as described in Example 1 (a, nucleotides 2399-3102 in SEQ ID N0: 1) was radioactively labeled with a-32P-dCTP using a random-primed labeling kit (Boehringer Mannheim) in accordance with the manufacturer's instructions. The radioactive probe, which had been denatured by heating, was hybridized in QuickHyb solution (from Strategene) at 68°C for one hour with a multiple-tissue Northern blot (10 ~g each of total RNA from rat brain, rat liver, rat lung, rat heart, rat kidney, rat testis, rat muscle and rat intestine, isolated as described by "Chomczinski and Sacchi, Anal. Biochem., 162, 156-159, 1987"); the blot was then washed, at 60°C, with O.lx SSC. After three days of exposure, a strong hybridization signal at about 5-6 kb was observed on brain RNA, while a substantially weaker signal of smaller size was observed on testis RNA and no signal was observed in any of the other tissues investigated (see Figure la).
Example 4 Expressing the mRNA for the novel GABAH receptor component in rat brain Unless otherwise described, the in-situ hybridization was carried out as described in "Molecular Neurobiology: A Practical ' 26 Approach. J. Chad and H. Wheat, eds. (Oxford: IRL Press), pp.205-225".
Two antisense oligonucleotides (reverse-complementary to nucleotides 2463-2498 and nucleotides 2538-2573, respectively, in SEQ ID N0: 1) were radioactively labeled with a-35S-dATP using terminal deoxynucleotide transferase (Boehringer Mannheim) in accordance with the manufacturer's instructions. These radioactive probes were applied to horizontal rat brain sections of approximately 15 Eun thickness and hybridized at 42°C for 16 hours in 4x SSC, 50% formamide. After that, the sections were washed at 55°C for 30 minutes in lx SSC and then exposed for 8 days. The autoradiograms for the two oligonucleotides gave a consistent picture (see Figure lb). The strongest signal was found in Purkinje cells of the cerebellum, while strong signals were also found in the cortex, the hippocampus and various thalamic nuclei, and weaker signals were found in the granular cells of the cerebellum.
Example 5 Cloning and sequencing the cDNA for the human form of the novel GABAB receptor component SEQ ID N0: 3 (DNA sequence) and SEQ ID
NO: 4 (amino acid sequence) cDNA was synthesized from 5 ~,g of total RNA from the whole brain of a 57-year-old man (from Clontech) using Superscript Reverse transcriptase (Gibco BRL) in accordance with the manufacturer's instructions. Several oligonucleotide primers, which were deduced from SEQ ID NO: 1 or from human EST sequences contained in the EMBL database, were used to amplify specific products from the human cDNA in PCR reactions. The primer pairs used in this case were as follows:
GBls/GB6as GBlSs(hs)/GBlBas GBl7s/GBllas GBl7s/GBl6as(hs) GB25s/GB4as GB25s/GB23as(hs) GB25sXbaI/GB23as(hs) GB22s(hs)/GBl6as(hs) GBls: 5'-CAGATCCGCAACGAGTCACTCCTG-3' GB2as: 5'-CAGGAGTGACTCGTTGCGGATCTG-3' GB3s: 5'-CAGTTTGACCAGAATATGGCAGC-3' GB4as: 5'-GCTGCCATATTCTGGTCAAACTG-3' GB6as: 5'-GACCTTCACCTCTCTGCTGTCTTG-3' GBllas: 5'-GAAGGAGGGTGGTACGTGTCTGTG-3' GBlSs(hs): 5'-CTACGATGGCATCTGGGTCATC-3' GBl6as(hs): 5'-GTCCCATTTCCGTTCCTCTTC-3' GBl7s: 5'-CTCAACGACAGCAAGTACATC-3' GBl8as: 5'-GATGTACTTGCTGTCGTTGAG-3' GBl9as(hs): 5'-GCTCTAGACCGTATTTTATTGCATCGTAG-3' GB22s(hs): 5'-GCGAATTCACAAAAAGACAAGACCATCATCCTG-3' GB23as(hs): 5'-GCGAATTCAGGATGGTGAGGGCAGAGAGGATG-3' GB25s: 5'-GTGAATTCGCGGCGCGGCATGGCTTC-3' GB25sXbaI: 5'-GTTCTAGACGCGGCGCGGCATGGCTTC-3' GB27as: 5'-CTGGTCCCGGGTCAGGAAGGAGAC-3' The PCR products were sequenced directly using the primers which had already been used for the amplification or using the abovementioned primers. New primers were deduced from the resulting sequences and then used for the PCR reactions and sequencings (see above for the list of the primers). It was finally possible to assemble the sequence SEQ ID N0: 3, which contains the open reading frame for a protein having the amino acid sequence SEQ ID NO: 4, from the individual sequences of the resulting PCR products.
After repeated sequencing from both directions, a G signal was also detected, in addition to the A signal, at positions 360 and 2605 in the sequence SEQ ID N0: 3, which means that the sequence reads either A or G at this point. In the case of position 360, this change as compared with SEQ ID NO: 3 would not lead to any change in the SEQ ID N0: 4 amino acid sequence; at position 2605, it would lead to a replacement of threonine with alanine at position 869 of the SEQ ID N0:4 sequence. Base pairs 1 to 8 in SEQ ID NO: 3 were stipulated by the primer GB25s, which was deduced from SEQ ID N0: 1. The possibility cannot be ruled out, therefore, that one or more positions in positions 1 to 8 in SEQ
ID N0: 3 is/are different from those indicated. Consequently, one or more of amino acids 1 to 3 in SEQ ID N0: 4 may be different from those indicated.
The PCR product GB25sXbaI/GB23as(hs) was subjected to a restriction digestion with Xbal and BglII. The PCR product GB22s(hs)/GBl6as(hs) was subjected to a restriction digestion with BglII and XhoI. The two PCR products were cloned (pBS-hsGB) into a pBSIIKS(-)-vector (from Stratagene) which had been previously cut with XbaI and XhoI.

SEQUENCE LISTING
<110> BASF-LYNX Bioscience AG
<120> Neuer metabotroper GAGA-Rezeptor Komplex aus dem zentralen Nervensystem <130> OZ0091/000001 <140> PCT/EP/99/06742 <141> 1999-09-11 <150> DE 19841941.4 <151> 1998-09-14 <160> 4 <170> PatentIn Ver. 2.0 <210>1 <211>3288 <212>DNA

<213>Rattus norvegicus <220>
<221> CD5 <222> (118)..(2940) <400> 1 ccggggcccg tgccggcgcc attgcgcggg agccgcgggc aaagctcggc gccgggcggc 60 gggccgggcc aggccatgcg ggccgagtga gctggcgccc gcagcccgcg gcgcggc 117 atg get tcc cog ccg agc tcc ggg cag ccc cgg ccg ccg ccg ccg ccg 165 Met Ala Ser Pro Pro Ser Ser Gly Gln Pro Arg Pro Pro Pro Pro Pro ccg ccg ccc gcg cgc ctg ctg ctg ccc ctg ctg ctg tcg ctg ctg ctg 213 Pro Pro Pro AIa Arg Leu Leu Leu Pro Leu Leu Leu Ser Leu Leu Leu tgg ttg gcg ccc ggg gcc tgg ggc tgg acg cgg ggc gcc ccc cgg ccg 261 Trp Leu Ala Pro Gly Ala Trp Gly Trp Thr Arg Gly Ala Pro Arg Pro ccg ccc agc agc ccg ccg ctc tcc atc atg ggc ctc atg ccg ctc acc 309 Pro Pro Ser Ser Pro Pro Leu Ser Ile Met Gly Leu MeL Pro Leu Thr aag gag gtg gcc aag ggc agc atc ggg cgc ggc gtg ctc ccc gcc gtg 357 Lys Glu Val Ala Lys Gly Ser Ile Gly P.rg Gly Val Leu Pro Ala Val gag cta gcc atc gag cag atc cgc aac gag tca ctc ctg cgc ccc tac 405 GIu Leu Ala Ile Glu Gln Ile Arg Asn Glu Ser Leu Leu Arg Pro Tyr ttc ctg gac ctg cga ctc tat gac acc gag tgt gac aat gca aag gga 453 Phe Leu Asp Leu Arg Leu Tyr Asp Thr Glu Cys Asp Asn Ala Lys Gly ctg aaa gcc ttc tat gac gca ata aag tat ggg ccg aac cat ttg atg 501 Leu Lys A1a Phe Tyr Asp Ala Ile Lys Tyr Gly Pro Asn His Leu Met gtg ttt gga ggc gtc tgt ccg tct gtc aca tct att atc gcg gag tcc 549 Val Phe Gly Gly Val Cys Pro Ser Val Thr Ser Ile Ile Ala Glu Sex ctc caa ggc tgg aat ctg gtg cag ctt tcc ttc gcc gcc acc acg cct 597 Leu Gln Gly Trp Asn Leu Val Gln Leu Ser Phe Ala Ala Thr Thr Pro gtt ctt gcg gat aag aag aag tac ccg tat ttc ttc cgg acg gtg ccg 645 Val Leu Ala Asp Lys Lys Lys Tyr Pro Tyr Phe Phe Arg Thr Val Pro tca gac aac gcg gtg aac ccc gcc atc ctg aag ctc ctg aag cac ttc &93 Ser Asp Asn Ala Val Asn Pro Ala Ile Leu Lys Leu Leu Lys His Phe cgc tgg cgg cgt gtg ggc aca ctc acg cag gac gtg cag cgc ttc tcc 741 Arg Trp Arg Arg Val Gly Thr Leu Thr Gln Asp Val Gln Arg Phe Ser gag gtg agg aat gac ctg act ggg gtt ctg tat ggg gaa gat att gag 789 Glu Val Arg Asn Asp Leu Thr Gly Val Leu Tyr Gly Glu Asp Ile Glu atc tca gac aca gag agc ttc tcc aat gat ccc tgc acc agc gtc aaa 837 Ile Ser Asp Thr Glu Ser Phe Ser Asn Asp Pro Cys Thr Ser Val Lys aag ctc aag ggg aat gac gtg cgg atc atc ctt ggc cag ttt gac cag 885 Lys Leu Lys Gly Asn Asp Va1 Arg Ile Ile Leu Gly Gln Phe Asp Gln aat atg gca gca aaa gtc ttc tgt tgt gcc ttc gag gag agc atg ttt 933 Asn Met Ala Ala Lys Val Phe Cys Cys Ala Phe Glu Glu Ser Met Phe ggc agc aag tac cag tgg atc atc ccg gga tgg tac gag cct gcg tgg 981 Gly Ser Lys Tyr Gln Trp IIe Ile Pro Gly Trp Tyr Glu Pro Ala Trp tgg gag cag gtg cat gtg gag gcc aat tcc tca cgc tgc ctg cgc aga 1029 Trp G1u Gln Val His Val Glu Ala Asn Ser Ser Arg Cys Leu Arg Arg agc ctc ctg get gcc atg gaa ggt tac atc gga gtg gac ttt gag ccc 1077 Ser Leu Leu Ala Ala Met Glu Gly Tyr Ile Gly Val Asp Phe Glu Pro ctg agc tcc aaa caa atc aag acc att tca ggg aag act cca cag cag 1125 Leu Ser Ser Lys Gln Ile Lys Thr Ile Ser Gly Lys Thr Pro Gln Gln tat gaa aga gag tac aac agc aaa cgt tca ggc gtg ggg ccc agc aag 1173 Tyr Glu Arg Glu Tyr Asn Ser Lys Arg Ser Gly Val Gly Pro Ser Lys ttc cat ggg tac gcc tac gat ggg atc tgg gtc atc gcc aag acc cta 1221 Phe His Gly Tyr Ala Tyr Asp Gly Ile Trp Val Ile Ala Lys Thr Leu cag agg gcc atg gag aca ctg cat gcc agt agc agg cac cag cgg atc 1269 Gln Arg Ala Met Glu Thr Leu His Ala Ser Ser Arg His Gln Arg Ile cag gac ttc aac tac aca gac cac acg ctg ggc aaa atc atc ctc aat 1317 Gln Asp Phe Asn Tyr Thr Asp His Thr Leu Gly Lys Ile Ile Leu Asn gcc atg aac gag acc aac ttc ttc ggg gtc acg ggt caa gtt gtg ttc 1365 Ala Met Asn Glu Thr Asn Phe Phe Gly Val Thr Gly Gln Val Val Phe cgg aac ggg gag aga atg gga acc att aaa ttt act caa ttt caa gac 1413 Arg Asn Gly Glu Arg Met Gly Thr Ile Lys Phe Thr Gln Phe Gln Asp agc aga gag gtg aag gtc ggc gaa tac aac gcg gtg get gac aca ctg 1461 Ser Arg Glu Val Lys Val Gly Glu Tyr Asn Ala Val Ala Asp Thr Leu gag atc atc aat gac acc ata agg ttc cag ggg tcc gag cca ccc aag 1509 Glu Ile Ile Asn Asp Thr Ile Arg Phe Gln Gly Ser Glu Pro Pro Lys gac aag acc atc att ctg gag cag ctt cgg aag atc tcg ctt cca ctg 1557 Asp Lys Thr Ile Ile Leu Glu Gln Leu Arg Lys Ile Ser Leu Pro Leu tat agc atc ctg tcc get ctc acc atc ctc ggc atg atc atg gcc agc 1605 Tyr Ser Ile Leu Ser Ala Leu Thr Ile Leu Gly Met Ile Met Ala Ser gcc ttc ctc ttc ttc aac atc aag aac cgg aac caa aag ctg att aag 1653 Ala Phe Leu Phe Phe Asn Ile Lys Asn Arg Asn Gln Lys Leu Ile Lys atg tca agc ccc tac atg aac aac ctc atc atc ctg gga gga atg ctg 1701 Met Ser Ser Pro Tyr Met Asn Asn Leu Ile Ile Leu Gly Gly Met Leu tcc tat gca tcc atc ttc ctc ttt ggc ctc gat ggg tcc ttc gtc tca 1749 Ser Tyr Ala Ser Ile Phe Leu Phe Gly Leu Asp Gly Ser Phe Va1 Ser gaa aag acc ttt gaa aca ctc tgc acg gtc cgg acc tgg att ctc acc 1797 Glu Lys Thr Phe Glu Thr Leu Cys Thr Val Arg Thr Trp Ile Leu Thr gtg ggc tac aca act gcc ttt ggg gcc atg ttt gca aag acc tgg agg 1845 Val Gly Tyr Thr Thr Ala Phe Gly Ala Met Phe Ala Lys Thr Trp Arg gtc cat gcc atc ttc aaa aat gtg aag atg aag aag aag atc atc aaa 1893 Val his Ala Ile Phe Lys Asn Val Lys Met Lys Lys Lys Ile Ile Lys gac cag aag ctg ctt gtg att gtg ggg ggc atg ctg ctc atc gac ctg 1941 Asp Gln Lys Leu Leu Val Ile Val Gly Gly Met Leu Leu Ile Asp Leu tgc atc ctg atc tgt tgg cag get gtg gac ccc ctg cgg agg aca gtg 1989 Cys Ile Leu Ile Cys Trp Gln Ala Val Asp Pro Leu Arg Arg Thr Val gag agg tac agc atg gag ccg gac cca gca ggc cgg gac atc tcc atc 2037 G1u Arg Tyr Ser Met Glu Pro Asp Pro Ala Gly Arg Asp Ile Ser Ile cgc cca ttg ctg gaa cac tgc gaa aac acc cac atg acc atc tgg ctt 2085 Arg Pro Leu Leu Glu His Cys Giu Asn Thr His Met Thr Ile Trp Leu ggc att gtc tac gcc tac aag ggg ctc ctc atg cta ttc ggt tgt ttc 2133 Gly Ile Val Tyr Ala Tyr Lys Gly Leu Leu Met Leu Phe Gly Cys Phe ttg gca tgg gaa acc cgc aat gtg agc atc cct gcc ctc aac gac agc 2181 Leu Ala Trp Glu Thr Arg Asn Val Ser Ile Pro Ala Leu Asn Asp Ser aag tac atc ggc atg agt gtg tac aat gtg ggg atc atg tgc atc atc 2229 Lys Tyr Ile Gly Met Ser Val Tyr Asn Val Gly Ile Met Cys Ile Ile ggg get get gtc tcc ttc ctg acg cgt gac cag ccc aac gtg cag ttc 2277 Gly Ala Ala Val Ser Phe Leu Thr Arg Asp Gln Pro Asn Val Gln Phe tgc atc gtg gcc ctg gtc atc atc ttc tgc agc acc atc act ctc tgc 2325 Cys Ile Val Ala Leu Val Ile Ile Phe Cys Ser Thr Ile Thr Leu Cys ctg gtg ttt gtg cca aag ctc atc act ctg agg aca aac cct gac gca 2373 Leu Val Phe Val Pro Lys Leu Ile Thr Leu Arg Thr Asn Pro Asp Ala gcc act cag aac agg cgg ttc cag ttc aca cag aac cag aag aaa gaa 2421 Ala Thr Gln Asn Arg Arg Phe Gln Phe Thr Gln Asn Gln Lys Lys Glu gat tcg aag acc tcc act tca gtc acc agc gtg aac cag gcg agc acg 2469 Asp Ser Lys Thr Ser Thr Ser Val Thr Ser Val Asn Gln Ala Ser Thr tca cgc ctg gag gga ctg cag tca gaa aac cac cgc ctt cga atg aag 2517 Ser Arg Leu Glu Gly Leu Gln Ser Glu Asn His Arg Leu Arg Met Lys atc aca gag ctg gac aaa gac ttg gaa gaa gtc acc atg cag cta caa 2565 Ile Thr Glu Leu Asp Lys Asp Leu Glu Glu Val Thr Met Gln Leu Gln gac aca cca gag aag acc aca tac atc aaa cag aat cac tac caa gag 2613 Asp Thr Pro Glu Lys Thr Thr Tyr Ile Lys Gln Asn His Tyr Gln Glu ctc aac gac atc ctc agc ttg ggc aac ttc aca gag agc aca gat gga 2661 Leu Asn Asp Ile Leu Ser Leu Gly Asn Phe Thr Glu Ser Thr Asp Gly 835 840 g45 gga aag gcc att cta aaa aat cac ctc gat caa aac ccc cag ctc cag 2709 Gly Lys Ala Ile Leu Lys Asn His Leu Asp Gln Asn Pro Gln Leu Gln tgg aac acg aca gag ccc tca aga aca tgc aaa gac ccc ata gaa gac 2757 Trp Asn Thr Thr Glu Pro Ser Arg Thr Cys Lys Asp Pro Ile Glu Asp atc aac tcc ccg gag cac atc cag cgc cgg ctg tcg ctc cag ctc ccc 2805 Ile Asn Ser Pro Glu His Ile Gln Arg Arg Leu Ser Leu Gln Leu Pro atc ctt cac cac gcc tac ctc cca tcc atc gga ggc gtg gat gcc agc 2853 Ile Leu His His Ala Tyr Leu Pro Ser Ile Gly Gly Val Asp Ala Ser tgc gtc agc ccc tgt gtc agc cct acc gcc agc cct cgc cac aga cac 2901 Cys Val Ser Pro Cys Val Ser Pro Thr Ala Ser Pro Arg His Arg His gta cca ccc tcc ttc cga gtc atg gtc tcg ggc ctg tag gggtgggagg 2950 Val Pro Pro Ser Phe Arg Val Met Val Ser Gly Leu cctggggccg gggcctcccc gggacagcac catgctgggc caaggcgcct gccacaggca 3010 cactgacggc ggcgagaagc tgggcaccat gctgcctctc cagactgctg gaatggcgct 3070 caggcagagc gggactcggc accgacctcg agccttatct gtgaaggtct tactctcaca 3130 gaggagagga atgacaatga cttctccttc ttggcgtctg caaacaaaga ggagttggga 3190 tgtctgaaac ttgcaaaaac aaatcaaact ctagacaaag gagagaggcc tcggactcct 3250 gctgtcctcg ccaagtggcc agagcaaggg ctctgcag 3288 <210> 2 <211> 940 <212> PRT
<213> Rattus norvegicus <400> 2 Met Ala Ser Pro Pro Ser Ser Gly Gln Pro Arg Pro Pro Pro Pro Pro Pro Pro Pro Ala Arg Leu Leu Leu Pro Leu Leu Leu Ser Leu Leu Leu Trp Leu Ala Pro Gly Ala Trp Gly Trp Thr Arg Gly Ala Pro Arg Pro Pro Pro Ser Ser Pro Pro Leu Ser Ile Met Gly Leu Met Pro Leu Thr Lys Glu Val Ala Lys Gly Ser Ile Gly Arg Gly Val Leu Pro Ala Va1 Glu Leu Ala Ile Glu Gln Ile Arg Asn Glu Ser Leu Leu Arg Pro Tyr Phe Leu Asp Leu Arg Leu Tyr Asp,Thr Glu Cys Asp Asn Ala Lys Gly Leu Lys A1a Phe Tyr Asp Ala Ile Lys Tyr Gly Pro Asn His Leu Met Val Phe Gly Gly Val Cys Pro Ser Val Thr Ser Ile Ile Ala Glu Ser 130 135 , 140 Leu Gln Gly Trp Asn Leu Val Gln Leu Ser Phe Ala Ala Thr Thr Pro Val Leu Ala Asp Lys Lys Lys Tyr Pro Tyr Phe Phe Arg Thr Val Pro Ser Asp Asn Ala Val Asn Pro Ala Ile Leu Lys Leu Leu Lys His Phe Arg Trp Arg Arg Va1 Gly Thr Leu Thr Gln Asp Val Gln Arg Phe Ser Glu Val Arg Asn Asp Leu Thr Gly Val Leu Tyr Gly Glu Asp Ile Glu Ile Ser Asp Thr Glu Ser Phe Ser Asn Asp Pro Cys Thr Ser Val Lys Lys Leu Lys Gly Asn Asp Val Arg Ile Ile Leu Gly Gln Phe Asp Gln Asn Met Ala Ala Lys Val Phe Cys Cys Ala Phe Glu Glu Ser Met Phe Gly Ser Lys Tyr Gln Trp Ile Ile Pro Gly Trp Tyr Glu Pro Ala Trp Trp Glu Gln Val His Val Glu Ala Asn Ser Ser Arg Cys Leu Arg Arg Ser Leu Leu Ala Ala Met Glu Gly Tyr Ile Gly Val Asp Phe Glu Pro Leu Ser Ser Lys Gln Ile Lys Thr Ile Ser Gly Lys Thr Pro Gln Gln Tyr Glu Arg Glu Tyr Asn Ser Lys Arg Ser Gly Val Gly Pro Ser Lys Phe His Gly Tyr Ala Tyr Asp Gly Ile Trp Val Ile Ala Lys Thr Leu Gln Arg Ala Met Glu Thr Leu His Ala Ser Ser Arg His Gln Arg Ile Gln Asp Phe Asn Tyr Thr Asp His Thr Leu Gly Lys Ile Ile Leu Asn Ala Met Asn Glu Thr Asn Phe Phe Gly Val Thr Gly Gln Val Val Phe Arg Asn Gly Glu Arg Met Gly Thr Ile Lys Phe Thr Gln Phe Gln Asp Ser Arg Glu Val Lys Val Gly Glu Tyr Asn Ala Val Ala Asp Thr Leu Glu Ile Ile Asn Asp Thr Ile Arg Phe Gln Gly Ser Glu Pro Pro Lys Asp Lys Thr Ile Ile Leu Glu Gln Leu Arg Lys Ile Ser Leu Pro Leu Tyr Ser Ile Leu Ser Ala Leu Thr Ile Leu Gly Met Ile Met Ala Ser Ala Phe Leu Phe Phe Asn Ile Lys Asn Arg Asn Gln Lys Leu Ile Lys Met Ser Ser Pro Tyr Met Asn Asn Leu Ile Ile Leu Gly Gly Met Leu Ser Tyr Ala Ser Ile Phe Leu Phe Gly Leu Asp Gly Ser Phe Val Ser G1u Lys Thr Phe Glu Thr Leu Cys Thr Val Arg Thr Trp Ile Leu Thr Val Gly Tyr Thr Thr Ala Phe Gly Ala Met Phe Ala Lys Thr Trp Arg Val His Ala Ile Phe Lys Asn Val Lys Met Lys Lys Lys Ile Ile Lys Asp Gln Lys Leu Leu Val I1e Val Gly Gly Met Leu Leu Ile Asp Leu Cys Ile Leu Ile Cys Trp G1n Ala Va1 Asp Pro Leu Arg Arg Thr Val Glu Arg Tyr Ser Met Glu Pro Asp Pro Ala Gly Arg Asp Ile Ser Ile Arg Pro Leu Leu Glu His Cys Glu Asn Thr His Met Thr Ile Trp Leu Gly Ile Val Tyr Ala Tyr Lys Gly Leu Leu Met Leu Phe Gly Cys Phe Leu Ala Trp Glu Thr Arg Asn Val Ser Ile Pro Ala Leu Asn Asp Ser Lys Tyr Ile Gly Met Ser Val Tyr Asn Val Gly Ile Met Cys Ile Ile Gly Ala Ala Val Ser Phe Leu Thr Arg Asp Gln Pro Asn Val Gln Phe Cys Ile Val Ala Leu Val Ile Ile Phe Cys Ser Thr Ile Thr Leu Cys Leu Val Phe Val Pro Lys Leu Ile Thr Leu Arg Thr Asn Pro Asp Ala Ala Thr Gln Asn Arg Arg Phe Gln Phe Thr Gln Asn Gln Lys Lys Glu Asp Ser Lys Thr Ser Thr Ser Val Thr Ser Val Asn Gln Ala Ser Thr Ser Arg Leu Glu Gly Leu Gln Ser Glu Asn His Arg Leu Arg Met Lys Ile Thr Glu Leu Asp Lys Asp Leu Glu Glu Va1 Thr Met Gln Leu Gln Asp Thr Pro Glu Lys Thr Thr Tyr Ile Lys Gln Asn His Tyr Gln Glu Leu Asn Asp I1e Leu Ser Leu Gly Asn Phe Thr Glu Ser Thr Asp Gly Gly Lys Ala Ile Leu Lys Asn His Leu Asp Gln Asn Pro Gln Leu Gln 850 ' 855 860 Trp Asn Thr Thr Glu Pro Ser Arg Thr Cys Lys Asp Pro Ile Glu Asp Ile Asn Ser Pro Glu His Ile Gln Arg Arg Leu Ser Leu Gln Leu Pro Ile Leu His His Ala Tyr Leu Pro Ser Ile Gly Gly Val Asp Ala Ser Cys Val Ser Pro Cys Val Ser Pro Thr Ala Ser Pro Arg His Arg His Val Pro Pro Ser Phe Arg Val Met Val Ser Gly Leu <210> 3 <211> 2826 <212> DNA
<213> Homo sapiens <220> ' <221> CDS
<222> (1)..(2826) <400> 3 atg get tcc ccg cgg agc tcc ggg cag ccc ggg ccg ccg ccg ccg ccg 48 Met Ala Ser Pro Arg Ser Ser Gly Gln Pro Gly Pro Pro Pro Pro Pro cca ccg ccg ccc gcg cgc ctg cta ctg cta ctg ctg ctg ccg ctg ctg 96 Pro Pro Pro Pro Ala Arg Leu Leu Leu Leu Leu Leu Leu Pro Leu Leu ctg cct ctg gcg ccc ggg gcc tgg ggc tgg gcg cgg ggc gcc ccc cgg 144 Leu Pro Leu Ala Pro Gly Ala Trp Gly Trp Ala Arg Gly Ala Pro Arg ccg ccg ccc agc agc ccg ccg ctc tcc atc atg ggc ctc atg ccg ctc 192 Pro Pro Pro Ser Ser Pro Pro Leu Ser Ile Met Gly Leu Met Pro Leu acc aag gag gtg gcc aag ggc agc atc ggg cgc ggt gtg ctc ccc gcc 240 Thr Lys Glu Val Ala Lys Gly Ser Ile Gly Arg Gly Val Leu Pro Ala gtg gaa ctg gcc atc gag cag atc cgc aac gag tca ctc ctg cgc ccc 288 Val Glu Leu Ala Ile Glu Gln Ile Arg Asn Glu Ser Leu Leu Arg Pro tac ttc ctc gac ctg cgg ctc tat gac acg gag tgc gac aac gca aaa 336 Tyr Phe Leu Asp Leu Arg Leu Tyr Asp Thr Glu Cys Asp Asn Ala Lys ggg ttg aaa gcc ttc tac gat gca ata aaa tac ggg ccg aac cac ttg 384 Gly Leu Lys Ala Phe Tyr Asp Ala Ile Lys Tyr Gly Pro Asn His Leu atg gtg ttt gga ggc gtc tgt cca tcc gtc aca tcc atc att gca gag 432 Met Val Phe Gly Gly Val Cys Pro Ser Val Thr Ser Ile Ile Ala Glu ~cc ctc caa ggc tgg aat ctg gtg cag ctt tct ttt get gca acc acg 480 5er Leu Gln Gly Trp Asn Leu Val Gln Leu Ser Phe Ala Ala Thr Thr cct gtt cta gcc gat aag aaa aaa tac cct tat ttc ttt cgg acc gtc 528 Pro Val Leu Ala Asp Lys Lys Lys Tyr Pro Tyr Phe Phe Arg Thr Val cca tca gac aat gcg gtg aat cca gcc att ctg aag ttg ctc aag cac 576 Pro Ser Asp Asn Ala Val Asn Pro Ala Ile Leu Lys Leu Leu Lys His tac cag tgg aag cgc gtg ggc acg ctg acg caa gac gtt cag agg ttc 624 Tyr Gln Trp Lys Arg Val Gly Thr Leu Thr G1n Asp Val Gln Arg Phe tct gag gtg cgg aat gac ctg act gga gtt ctg tat ggc gag gac att 672 Ser Glu Val Arg Asn Asp Leu Thr Gly Val Leu Tyr Gly Glu Asp Ile gag att tca gac acc gag agc ttc tcc aac gat ccc tgt acc agt gtc 720 Glu Ile Ser Asp Thr Glu Ser Phe Ser Asn Asp Pro Cys Thr Ser Val aaa aag ctg aag ggg aat gat gtg cgg atc atc ctt ggc cag ttt gac 768 Lys Lys Leu Lys Gly Asn Asp Val Arg Ile Ile Leu Gly Gln Phe Asp cag aat atg gca gca aaa gtg ttc tgt tgt gca tac gag gag aac atg 816 Gln Asn Met Ala Ala Lys Val Phe Cys Cys A1a Tyr Glu Glu Asn Met tat ggt agt aaa tat cag tgg atc att ccg ggc tgg tac gag cct tct 864 Tyr Gly Ser Lys Tyr Gln Trp Ile Ile Pro Gly Trp Tyr Glu Pro Ser tgg tgg gag cag gtg cac acg gaa gcc aac tca tcc cgc tgc ctc cgg 912 Trp Trp Glu Gln Val His Thr G1u Ala Asn Ser Ser Arg Cys Leu Arg aag aat ctg ctt get gcc atg gag ggc tac att ggc gtg gat ttc gag 960 Lys Asn Leu Leu Ala Ala Met Glu Gly Tyr Ile Gly Val Asp Phe Glu ccc ctg agc tcc aag cag atc aag acc atc tca gga aag act cca cag 1008 Pro Leu Ser Ser Lys Gln Ile Lys Thr Ile Ser Gly Lys Thr Pro Gln cag tat gag aga gag tac aac aac aag cgg tca ggc gtg ggg ccc agc 1056 Gln Tyr Glu Arg G1u Tyr Asn Asn Lys Arg Ser Gly Val Gly Pro Ser aag ttc cac ggg tac gcc tac gat ggc atc tgg gtc atc gcc aag aca 1104 Lys Phe His Gly Tyr Ala Tyr Asp Gly Ile Trp Val Ile Ala Lys Thr ctg cag agg gcc atg gag aca ctg cat gcc agc agc cgg cac cag cgg 1152 Leu Gln Arg Ala Met Glu Thr Leu His Ala Ser Ser Arg His Gln Arg atc cag gac ttc aac tac acg gac cac acg ctg ggc agg atc atc ctc 1200 Ile Gln Asp Phe Asn Tyr Thr Asp His Thr Leu Gly Arg Ile Ile Leu aat gcc atg aac gag acc aac ttc ttc ggg gtc acg ggt caa gtt gta 1248 Asn Ala Met Asn Glu Thr Asn Phe Phe Gly Val Thr Gly Gln Val Val ttc cgg aat ggg gag aga atg ggg acc att aaa ttt act caa ttt caa 1296 Phe Arg Asn Gly G1u Arg Met Gly Thr Ile Lys Phe Thr Gln Phe Gln gac agc agg gag gtg aag gtg gga gag tac aac get gtg gcc gac aca 1349 Asp Ser Arg Glu Val Lys Val Gly Glu Tyr Asn Ala Val Ala Asp Thr ctg gag atc atc aat gac acc atc agg ttc caa gga tcc gaa cca cca 1392 Leu G1u Ile Ile Asn Asp Thr Ile Arg Phe Gln Gly Ser Glu Pro Pro aaa gac aag acc atc atc ctg gag cag ctg cgg aag atc tcc cta cct 1440 Lys Asp Lys Thr Ile Ile Leu Glu Gln Leu Arg Lys Ile Ser Leu Pro ctc tac agc atc ctc tct gcc ctc acc atc ctc ggg atg atc atg gcc 1488 Leu Tyr Ser Ile Leu Ser Ala Leu Thr Ile Leu Gly Met Ile Met Ala agt get ttt ctc ttc ttc aac atc aag aac cgg aat cag aag ctc ata 1536 Ser Ala Phe Leu Phe Phe Asn Ile Lys Asn Arg Asn Gln Lys Leu Ile __. 500 505 510 aag atg tcg agt cca tac atg aac aac ctt atc atc ctt gga ggg atg 1584 Lys Met Ser Ser Pro Tyr Met Asn Asn Leu Ile Ile Leu Gly Gly Met ctc tcc tat get tcc ata ttt ctc ttt ggc ctt gat gga tcc ttt gtc 1632 Leu Ser Tyr Ala Ser Ile Phe Leu Phe Gly Leu Asp Gly Ser Phe Val tct gaa aag acc ttt gaa aca ctt tgc acc gtc agg acc tgg att ctc 1680 Ser G1u Lys Thr Phe Glu Thr Leu Cys Thr Val Arg Thr Trp Ile Leu acc gtg ggc tac acg acc get ttt ggg gcc atg ttt gca aag acc tgg 1728 Thr Val Gly Tyr Thr Thr Ala Phe Gly Ala Met Phe Ala Lys Thr Trp aga gtc cac gcc atc ttc aaa aat gtg aaa atg aag aag aag atc atc 1776 Arg Va1 His Ala Ile Phe Lys Asn Val Lys Met Lys Lys Lys Ile Ile aag gac cag aaa ctg ctt gtg atc gtg ggg ggc atg ctg ctg atc gac 1824 Lys Asp G1n Lys Leu Leu Val Ile Val Gly Gly Met Leu Leu Ile Asp ctg tgt atc ctg atc tgc tgg cag get gtg gac ccc ctg cga agg aca 1872 Leu Cys Ile Leu Ile Cys Trp Gln Ala Val Asp Pro Leu Arg Arg Thr gtg gag aag tac agc atg gag ccg gac cca gca gga cgg gat atc tcc 1920 Val Glu Lys Tyr Ser Met Glu Pro Asp Pro Ala Gly Arg Asp Ile Ser atc cgc cct ctc ctg gag cac tgt gag aac acc cat atg acc atc tgg 1968 Ile Arg Pro Leu Leu Glu His Cys Glu Asn Thr His Met Thr Ile Trp ctt ggc atc gtc tat gcc tac aag gga ctt ctc atg ttg ttc ggt tgt 2016 Leu Gly Ile Val Tyr Ala Tyr Lys Gly Leu Leu Met Leu Phe Gly Cys ttc tta get tgg gag acc cgc aac gtc agc atc ccc gca ctc aac gac 2069 Phe Leu Ala Trp Glu Thr Arg Asn Val Ser Ile Pro Ala Leu Asn Asp agc aag tac atc ggg atg agt gtc tac aac gtg ggg atc atg tgc atc 2112 Ser Lys Tyr Ile Gly Met Ser Val Tyr Asn Val Gly Ile Met Cys Ile atc ggg gcc get gtc tcc ttc ctg acc cgg gac cag ccc aat gtg cag 2160 Ile Gly A1a Ala Val Ser Phe Leu Thr Arg Asp Gln Pro Asn Val Gln ttc tgc atc gtg get ctg gtc atc atc ttc tgc agc acc atc acc ctc 2208 Phe Cys Ile Val Ala Leu Val Ile Ile Phe Cys Ser Thr Ile Thr Leu tgc ctg gta ttc gtg ccg aag ctc atc acc ctg aga aca aac cca gat 2256 Cys Leu Val Phe Val Pro Lys Leu Ile Thr Leu Arg Thr Asn Pro Asp gca gca acg cag aac agg cga ttc cag ttc act cag aat cag aag aaa 2304 Ala Ala Thr Gln Asn Arg Px g Phe Gln Phe Thr Gln Asn Gln Lys Lys gaa gat tct aaa acg tcc acc tcg gtc acc agt gtg aac caa gcc agc 2352 Glu Asp Ser Lys Thr Ser Thr Ser Val Thr Ser Val Asn Gln Ala Ser aca tcc cgc ctg gag ggc cta cag tca gaa aac cat cgc ctg cga atg 2900 Thr Ser Arg Leu Glu Gly Leu Gln Ser Glu Asn His Arg Leu Arg Met aag atc aca gag ctg gat aaa gac ttg gaa gag gtc acc atg cag ctg 2498 ' Lys Ile Thr Glu Leu Asp Lys Asp Leu Glu Glu Val Thr Met Gln Leu cag gac aca cca gaa aag acc acc tac att aaa cag aac cac tac caa 2496 Gln Asp Thr Pro Glu Lys Thr Thr Tyr Ile Lys Gln Asn His Tyr Gln gag ctc aat gac atc ctc aac ctg gga aac ttc act gag agc aca gat 2544 Glu Leu Asn Asp Ile Leu Asn Leu Gly Asn Phe Thr Glu Ser Thr Asp gga gga aag gcc att tta aaa aat cac ctc gat caa aat ccc cag cta 2592 Gly Gly Lys Ala Ile Leu Lys Asn His Leu Asp Gln Asn Pro Gln Leu cag tgg aac aca nca gag ccc tct cga aca tgc aaa gat cct ata gaa 2640 Gln Trp Asn Thr Xaa Glu Pro Ser Arg Thr Cys Lys Asp Pro Ile Glu gat ata aac tct cca gaa cac atc cag cgt cgg ctg tcc ctc cag ctc 2688 Asp Ile Asn Ser Pro Glu His Ile Gln Arg Arg Leu Ser Leu Gln Leu ccc atc ctc cac cac gcc tac ctc cca tcc atc gga ggc gtg gac gcc 2736 Pro Ile Leu His His Ala Tyr Leu Pro Ser Ile Gly Gly Val Asp Ala agc tgt gtc agc ccc tgc gtc agc ccc acc gcc agc ccc cgc cac aga 2784 Ser Cys Val Ser Pro Cys Val Ser Pro Thr Ala Ser Pro Arg His Arg cat gtg cca ccc tcc ttc cga gtc atg gtc tcg ggc ctg taa 2826 His Val Pro Pro Ser Phe Arg Val Met Val Ser Gly Leu <210> 4 <211> 941 <212> PRT
<213> Homo Sapiens <400> 4 Met Ala Ser Pro Arg Ser Ser Gly Gln Pro Gly Pro Pro Pro Pro Pro _ . 1 5 10 15 Pro Pro Pro Pro Ala Arg Leu Leu Leu Leu Leu Leu Leu Pro Leu Leu Leu Pro Leu Ala Pro Gly Ala Trp Gly Trp Ala Arg Gly Ala Pro Arg Pro Pro Pro Ser Ser Pro Pro Leu Ser Ile Met Gly Leu Met Pro Leu Thr Lys G1u Val Ala Lys Gly Ser Ile Gly Arg Gly Val Leu Pro Ala Val Glu Leu Ala Ile Glu Gln Ile Arg Asn Glu Ser Leu Leu Arg Pro Tyr Phe Leu Asp Leu Arg Leu Tyr Asp Thr Glu Cys Asp Asn Ala Lys Gly Leu Lys Ala Phe Tyr Asp Ala Ile Lys Tyr Gly Pro Asn His Leu Met Val Phe Gly Gly Val Cys Pro Ser Val Thr Ser Ile Ile Ala Glu Ser Leu Gln Gly Trp Asn Leu Val G1n Leu Ser Phe Ala Ala Thr Thr Pro Val Leu Ala Asp Lys Lys Lys Tyr Pro Tyr Phe Phe Arg Thr Val Pro Ser Asp Asn Ala Val Asn Pro Ala Ile Leu Lys Leu Leu Lys His Tyr Gln Trp Lys Arg Val Gly Thr Leu Thr Gln Asp Val Gln Arg Phe Ser Glu Val Arg Asn Asp Leu Thr Gly Va1 Leu Tyr Gly Glu Asp Ile Glu Ile Ser Asp Thr Glu Ser Phe Ser Asn Asp Pro Cys Thr Ser Val Lys Lys Leu Lys Gly Asn Asp Val Arg Ile Ile Leu Gly Gln Phe Asp Gln Asn Met Ala Ala Lys Val Phe Cys Cys Ala Tyr Glu Glu Asn Met Tyr Gly Ser Lys Tyr Gln Trp Ile Ile Pro Gly Trp Tyr Glu Pro Ser ' Trp Trp Glu Gln Val His Thr Glu Ala Asn Ser Ser Arg Cys Leu Arg Lys Asn Leu Leu Ala Ala Met Glu Gly Tyr Ile Gly Val Asp Phe Glu Pro Leu Ser Ser Lys Gln Ile Lys Thr Ile Ser Gly Lys Thr Pro Gln Gln Tyr Glu Arg Glu Tyr Asn Asn Lys Arg Ser Gly Val Gly Pro Ser Lys Phe His Gly Tyr Ala Tyr Asp Gly Ile Trp Val Ile Ala Lys Thr Leu Gln Arg Ala Met Glu Thr Leu His Ala Ser Ser Arg His Gln Arg Ile Gln Asp Phe Asn Tyr Thr Asp His Thr Leu G1y Arg Ile I1e Leu Asn Ala Met Asn Glu Thr Asn Phe Phe Gly Val Thr Gly Gln Val Val Phe Arg Asn Gly Glu Arg Met Gly Thr Ile Lys Phe Thr Gln Phe Gln Asp Ser Arg Glu Val Lys Val Gly Glu Tyr Asn Ala Val Ala Asp Thr Leu Glu Ile Ile Asn Asp T'hr Ile Arg Phe G1n Gly Ser Glu Pro Pro Lys Asp Lys Thr Ile Ile Leu Glu Gln Leu Arg Lys Ile Ser Leu Pro Leu Tyr Ser Ile Leu Ser Ala Leu Thr Ile Leu Gly Met Ile Met Ala Ser Ala Phe Leu Phe Phe Asn Ile Lys Asn Arg Asn Gln Lys Leu I1e Lys Met Ser Ser Pro Tyr Met Asn Asn Leu Ile Ile Leu Gly Gly Met Leu Ser Tyr Ala Ser Ile Phe Leu Phe Gly Leu Asp Gly Ser Fhe Val Ser Glu Lys Thr Phe Glu Thr Leu Cys Thr Val Arg Thr Trp Ile Leu Thr Val Gly Tyr Thr Thr Ala Phe Gly Ala Met Phe Ala Lys Thr Trp Arg Val His Ala Ile Phe Lys Asn Val Lys Met Lys Lys Lys Ile Ile Lys Asp Gln Lys Leu Leu Val Ile Val Gly Gly Met Leu Leu Ile Asp Leu Cys Ile Leu Ile Cys Trp Gln Ala Val Asp Pro Leu Arg Arg Thr Val G1u Lys Tyr Ser Mez Glu Pro Asp Pro Ala Gly Arg Asp Ile Ser Ile Arg Pro Leu Leu Glu His Cys Glu Asn Thr His Met Thr Ile Trp Leu Gly Ile Val Tyr A1a Tyr Lys Gly Leu Leu Met'Leu Phe Gly Cys Phe Leu Ala Trp Glu Thr Arg Asn Val Ser Ile Pro Ala Leu Asn Asp Ser Lys Tyr Ile Gly Met Ser Val Tyr Asn Val Gly Ile Met Cys Ile Ile Gly Ala Ala Val Ser Phe Leu Thr Arg Asp Gln Pro Asn Val Gln Phe Cys Ile Val Ala Leu Va1 Ile Ile Phe Cys Ser Thr Ile Thr Leu Cys Leu Val Phe Val Pro Lys Leu Ile Thr Leu Arg Thr Asn Pro Asp Ala Ala Thr Gln Asn Arg Arg Phe Gln Phe Thr Gln Asn Gln Lys Lys Glu Asp Ser Lys Thr Ser Thr Ser Val Thr Ser Val Asn Gln Ala Ser Thr Ser Arg Leu Glu Gly Leu Gln Ser Glu Asn His Arg Leu Arg Mez Lys Ile Thr Glu Leu Asp Lys Asp Leu Glu Glu Val Thr Met G1n Leu Gln Asp Thr Pro Glu Lys Thr Thr Tyr Ile Lys Gln Asn His Tyr Gln Glu Leu Asn Asp Ile Leu Asn Leu Gly Asn Phe Thr Glu Ser Thr Asp Gly Gly Lys A1a Ile Leu Lys Asn His Leu Asp Gln Asn Pro Gln Leu Gln Trp Asn Thr Xaa Glu Pro Ser Arg Thr Cys Lys Asp Pro Ile Glu Asp Ile Asn Ser Pro Glu His Ile Gln Arg Arg Leu Ser Leu Gln Leu Pro Ile Leu His His Ala Tyr Leu Pro Ser Ile Gly Gly Val Asp Ala Ser Cys Val Ser Pro Cys Va1 Ser Pro Thr Ala Ser Pro Arg His Arg His Val Pro Pro Ser.Phe Arg Val Met Val Ser Gly Leu

Claims

We claim:

1. A protein heteromer which contains at least one GABA B
receptor protein and at least one protein having the amino acid sequence depicted in SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence which can be obtained from this sequence by the substitution, inversion, insertion or deletion of one or more amino acid residues, with at least one of the essential biological properties of the protein depicted in SEQ ID No: 2 or SEQ ID NO: 4, or of the protein heteromer, still being conserved.

2. An isolated protein which contains the amino acid sequence depicted in SEQ ID NO: 2 or SEQ ID N0: 4, or a sequence which can be obtained from this sequence by the substitution, inversion, insertion or deletion of one or more amino acid residues, with at least one of the essential biological properties of the protein depicted in SEQ ID NO: 2 or SEQ ID
NO: 4 still being conserved.

3. A nucleic acid sequence which encodes a protein as claimed in claim 2.

4. A nucleic acid sequence as claimed in claim 3, which encodes a protein which possesses at least 60% identity with the sequence depicted in SEQ ID NO: 2 or SEQ ID NO: 4.

5. A nucleic acid sequence as claimed in claim 3, which contains the sequence depicted in SEQ ID NO: 1 or SEQ ID NO: 3.

6. A recombinant nucleic acid construct which contains a nucleic acid sequence as claimed in claim 3 or a nucleic acid sequence as claimed in claim 3 and a sequence which encodes a GABA B receptor protein, functionally linked to at least one genetic regulatory element.

7. A host organism which is transformed with a nucleic acid sequence as claimed in claim 3 or a recombinant nucleic acid construct as claimed in claim 6 or with a nucleic acid sequence as claimed in claim 3 or a recombinant nucleic acid construct as claimed in claim 6 together with a sequence which encodes a GABA B receptor protein.

8. A host organism which is transformed with a recombinant nucleic acid construct as claimed in claim 6.

9. A transgenic animal which contains a functional or non-functional nucleic acid sequence as claimed in claims 3 to 5 or a functional or non-functional nucleic acid construct as claimed in claim 6.

10. A transgenic animal in whose germ cells, or the entirety or a part of the somatic cells, or in whose germ cells and the entirety or a part of the somatic cells, the nucleotide sequence as claimed in claim 3 has been altered by recombinant methods or interrupted by inserting DNA elements.

11. The use of a nucleic acid sequence as claimed in claim 3, of a nucleic acid construct as claimed in claim 6, of a protein heteromer as claimed in claim 1 or of a protein as claimed in claim 2 for identifying proteins which exhibit specific binding affinities for a protein heteromer as claimed in claim 1 or a protein as claimed in claim 2, or for identifying nucleic acids which encode proteins which exhibit specific binding affinities for a protein heteromer as claimed in claim 1 or a protein as claimed in claim 2.

12. The use of the two-hybrid system or biochemical methods for identifying the interaction domains of metabotropic receptors, and their use for pharmacotherapeutic intervention.

13. The use of the information resulting from the elucidation of the structure of a protein heteromer as claimed in claim 1 or of a protein as claimed in claim 2 for selectively discovering or for selectively preparing substances having a specific binding affinity for a protein heteromer as claimed in claim 1 or a protein as claimed in claim 2.

14. The use of a protein heteromer as claimed in claim 1 or of a protein as claimed in claim 2, or peptide fragments thereof, as an antigen for producing specific monoclonal or polyclonal antibodies or antibody mixtures which are directed against proteins as claimed in claim 1 or 2.

15. A monoclonal or polyclonal antibody, or an antibody mixture, which specifically recognizes proteins as claimed in claim 1 or 2.

16. The use of a nucleic acid sequence as claimed in claim 3, or of a fragment thereof, for isolating a genomic sequence by way of homology screening.

17. The use of a nucleic acid sequence as claimed in claim 3 as a marker for human hereditary diseases.

18. The use of a nucleic acid sequence as claimed in claim 3, or parts thereof, for gene therapy.

19. The use of a nucleic acid sequence which is complementary to the nucleic acid sequence as claimed in claim 3, or to parts of this sequence, for gene therapy.

20. A process for discovering substances having a specific binding affinity for a protein as claimed in claim 1 or 2, which process comprises the following steps:
a) incubating the protein as claimed in claim 1 or 2 with the substance to be tested b) detecting the binding of the substance to be tested to the protein.

21. A process as claimed in claim 20, wherein the binding is detected by measuring the antagonization or agonization of the GABA B receptor activity.

22. A process as claimed in claim 20, wherein the binding of substances to a protein as claimed in claim 1 is detected by measuring a physiological effect, such as a change in the concentration of calcium, cAMP or IP3 or in the membrane potential.

23. A process for qualitatively or quantitatively detecting a nucleic acid as claimed in claim 3 in a biological sample, which process comprises one or more of the following steps:
a) incubating a biological sample with a known quantity of nucleic acid as claimed in claim 3 or a known quantity of oligonucleotides which are suitable for use as primers for amplifying the nucleic acid as claimed in claim 3, or mixtures thereof, b) detecting the nucleic acid as claimed in claim 3 by specific hybridization or PCR amplification, c) comparing the quantity of hybridizing nucleic acid as claimed in claim 3, or of nucleic acid as claimed in claim 3 which is obtained by PCR amplification, with a standard.

24. A process for qualitatively and quantitatively detecting a protein as claimed in claim 1 or 2 in a biological sample, which process comprises one or more of the following steps:
a) incubating a biological sample with an antibody as claimed in claim 15 which is specifically directed against proteins as claimed in claim 1 or 2, b) detecting the antibody/antigen complex, c) comparing the quantities of the antibody/antigen complex with a quantity standard.

25. A process for discovering substances which bind specifically to a protein having an amino acid sequence as claimed in claim 2, which process comprises one or more of the following steps:
a) expressing the protein in eukaryotic or prokaryotic cells, b) incubating the protein with the substances to be tested, c) detecting the binding of a substance to the receptor or detecting an effect on the receptor function.

26. A process for discovering substances which bind specifically to a protein having an amino acid sequence as claimed in claim 2, or to a nucleic acid sequence as claimed in claim 3, and thereby elicit inhibitory or activating functional effects on GABAergic signal transmission in central nervous neurones.

27. A process for discovering substances which inhibit or reinforce the interaction of proteins having amino acid sequences as claimed in claim 2 with other metabotropic receptors.

28. A process as claimed in claim 27, where the interaction takes place specifically with amino acid sequence 785-816 in SEQ ID
NO: 2 or 786-817 in SEQ ID N0: 4.

29. The use of substances which reduce or prevent the natural interaction of a metabotropic receptor with a protein of SEQ ID NO: 2 or SEQ ID NO: 4 for producing drugs for the treatment of disorders which can be beneficially influenced by modulation of the activity of metabotropic receptors.

30. The use as claimed in claim 29, wherein peptides or polypeptides are used as substances.

31. A process for discovering substances which inhibit or reinforce the interaction of ligands with the protein heteromer as claimed in claim 1 or proteins having amino acid sequences as claimed in claim 2.

32. A process for discovering substances which inhibit or reinforce the interaction of proteins having amino acid sequences as claimed in claim 2 with G proteins or other signal transduction molecules.

33. A process for qualitatively and quantitatively determining proteins as claimed in claim 2 using specific agonists or antagonists.

34. A process for quantifying the protein activity of a protein as claimed in claim 2.