AU6081799A - Metabotropic gaba receptor complex issued from the central nervous system - Google Patents

Description

Novel metabotropic GABA receptor complex from the central nervous system 5 The present invention relates to isolated proteins which encode novel metabotropic GABA receptors and which form a novel metabotropic GABA receptor complex (= protein heteromer) with GABAB receptor proteins, and to nucleic acid sequences or recombinant nucleic acid constructs which encode the proteins. 10 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 15 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 20 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 25 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 30 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 35 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 40 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]. 45 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 GABA 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 (= GABAB receptors) are also termed metabotropic GABA 5 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 10 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 GABAB receptors which regulate the release of a specific neurotransmitter or 15 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. GABAB receptors which activate various potassium channels by way of Gi proteins 20 are found at the post-synapse. In transgenic mice, null mutations for such a channel leads [sic] 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. GABAB receptor 25 agonists exhibit a positive effect in animal models for chronic pain and cocaine dependency. Antagonists have a positive effect in models of "absence epilepsy" (Bettler et al., Curr. Opin. Neurolbiol., 8, 1998: 345 - 350). Activation of GABAB receptors dampens overstimulated neuronal connections. GABAB receptors are 30 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, 35 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 40 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 45 in the peripheral nervous system (PNS), for example in association with inflammations and disorders of the respiratory tract. Substances which act on the GABAB receptor are particularly 3 suitable for the treatment of asthma, anaphylactic bronchospasm, airway hyperreactivity and pathological coughing. These substances can also have beneficial effects on functional disturbances or degenerative disorders of the retina such as, for 5 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 10 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, 15 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 20 GABAB receptor exhibits similarly high affinities for the known GABAB receptor antagonists as are found in the brain. By contrast, the affinities of GABAB 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). 25 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 GABAB receptors play a central role in various pathological 30 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 35 and characterize novel GABAB receptors or proteins which interact with the GABAB 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 40 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 GABAB receptor, are potential candidates for active compounds against diseases such as epilepsy, stroke, psychological 45 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 GABAB 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 5 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, 10 or with the isolated protein which contains 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 15 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 20 metabotropic GABA 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 GABAB receptor proteins which can advantageously be present in the protein heteromers are described in W097/46675. In W097/46675, 25 these GABAB receptor proteins have the sequence designations or clone names "SEQ ID NO:1" or "GABABRla rat" (cloned from Rattus norvegicus), "SEQ ID NO:3" or "GABABRla/b human" (cloned from Homo sapiens), "SEQ ID NO:5" or "GABABRlb rat" (cloned from Rattus norvegicus) and "SEQ ID NO:7" or "GABABRlb human" (cloned 30 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 35 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 still being conserved. 40 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 45 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 5 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 NO: 2 or SEQ ID NO: 4, as calculated using the "Altschul et al., J. Mol. Biol., 215, 403-410, 1990" algorithm. The essential biological property [sic] of the proteins or protein heteromers according to the invention are to be 10 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 15 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 20 with the abovementioned, known GABAB 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 25 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. 30 An important physiological property of native GABAB 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 NO: 2 or 4 according to the 35 invention with GIRK1 and GIRK2 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 40 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 GABA and leads to activation of GIRKs. The coupling of the GABAB 45 receptors with GIRKs therefore depends on the physical interac tion in the protein heteromer according to the invention.

6 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 5 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 [sic] receptor 1A or amino acids 785-816 in SEQ ID NO: 2 and ami no acids 786-817 in SEQ ID NO: 4) and have alpha-helical struc 10 tures (based on secondary structure predictions [Garnier et al., J. Mol. Biol., 120, 1978: 97-120]). The interaction between these domains mediates specifically for the formation of the heteromer, but not the formation of possibly homodimers or homomultimers. 15 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. 20 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 NO: 4. The nucleic acid sequences from Rattus norvegicus and Homo sapiens are depicted in SEQ ID NO: 1 and SEQ ID NO: 3, 25 respectively. The nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 3 according to the invention, or their functional equivalents such as allele variants, can be obtained following isolation and sequencing. 30 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 100% homology. Allele variants comprise, in particular, those functional variants which can be 35 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, 40 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 45 homologs of SEQ ID NO: 1 or SEQ ID NO: 3, for example their homologs from other mammals, truncated sequences or 7 single-stranded DNA or RNA of the coding and non-coding DNA sequences. Such functional equivalents can be isolated from other 5 vertebrates, such as mammals, using the DNA sequences described in SEQ No: 1 or SEQ ID NO: 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 10 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 GABA receptors, in 15 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 20 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 0 C lower than those for DNA:RNA hybrids of the same length. 25 Standard conditions are to be understood, for example, as being, depending on the nucleic acid, temperatures of from 42 to 58 0 C in an aqueous buffer solution having a concentration 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 0 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 0 C, preferably from about 30 0 C to 45 0 C. For DNA:RNA hybrids, the hybridization conditions are advantageously 0.1 x SSC and temperatures of from about 30 0 C to 55 0 C, preferably of from about 45 0 C to 55 0 C. These temperatures which are indicated 35 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 40 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 45 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. 5 Homologs of the sequences SEQ ID NO: 1 and SEQ ID NO: 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 10 nucleotide replacements, by (an) insertion(s) and/or (a) deletion(s) 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, 15 including those from organisms of another species. Derivatives are also advantageously to be understood as being variants whose nucleotide sequence [sic] have been altered in the -1 to -1000 region upstream of the start codon such that 20 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. 25 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 30 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 35 sequence which encodes a GABAB 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 40 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 45 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 9 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 5 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 10 be inserted at the 3' end of the nucleic acid sequences. The nucleic acid sequences for the sequences SEQ ID NO: 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. 15 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, 1-PR (sic] or 1-PL [sic] 20 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 SPO2, in the yeast promoters such as ADC1, MFa [sic], AC, P 60, CYC1 and GAPDH, or in mammalian promoters such as CaM-kinaseII, CMV, 25 nestin, L7, BDNF, NF, MBP, NSE, P-globin, GFAP, GAP43, tyrosine hydroxylase, kainate receptor subunit 1 and glutamate receptor subunit B. In principle, all natural promoters, together with their 30 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 35 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. 40 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 45 addition to this, however, it is also possible to reinforce translation by, for example, improving the stability of the mRNA.

10 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. 15 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. 20 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 25 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, baculovirus and adenovirus, 30 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. 35 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 40 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 45 ribosomes.

11 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 5 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 10 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 15 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 20 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 25 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 30 (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 35 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 40 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 45 COS, Hela, HEK293, Sf9 or CHO cells.

12 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 5 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 10 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 15 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. 20 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 25 phages, or transposons, and/or other advantageous regulatory sequences, form [sic] 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 and vectors such as pcDNA3neovector or HEK293 cells and CMV vector which are 30 suitable for mammalian cells [sic]. 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 35 and examples). Figures la and lb depict the analysis of the expression of the mRNA corresponding to SEQ ID NO: 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 GABAB receptor and indicates that the protein depicted in 40 SEQ ID NO: 2 or SEQ ID NO: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 NO: 2 is consequently an interesting target for understanding in relation to learning and memory and for developing new cognitive 45 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 NO:4 and their functional equivalents, 13 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 5 possessing the sequence SEQ ID NO:2 or SEQ ID NO: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 10 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 15 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 20 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 25 or proteins according to the invention consequently represent interesting targets for developing novel substances which [lacuna] for producing medicaments for treating diseases such as neurological disorders, such as epilepsy, stroke, psychological disorders, such as anxiety, manic-depressive disorders, migraine, 30 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 35 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, 40 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. 45 The gene for the already known GABAB receptor is located in the vicinity of the chromosomal locus which is associated with 14 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 NO: 1 and SEQ ID 5 NO: 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 10 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. 15 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 acid [sic] according to the invention, or parts thereof, can also be used 20 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 25 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 30 information about the (patho)physiology of the sequences according to the invention, either on their own or complexed with the GABAB receptor, can be generated by way of transgene overexpression or genetic mutation (null mutation or specific deletions, insertions or modifications) elicited by homologous 35 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. 40 The interaction of the known GABAB 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 45 regard to the abovementioned neurological and psychological disorders which are connected with the GABAB receptor. Low molecular weight effectors or peptides which exert a positive or 15 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 5 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 10 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, 15 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, 20 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. 25 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. 30 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. 35 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 40 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. 45 16 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 LD [sic] NO: 2 or 4. 5 Such substances preferentially bind to the following sequence re gions: (i) to amino acid sequence 887-921 of the GABA receptor 1A or 10 (ii) to amino acid sequence 785-816 of SEQ ID NO: 2 or (iii) to amino acid sequence 786-817 of SEQ ID NO: 4. 15 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 20 (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 25 comprises the following steps: a) incubating the protein(s) with the substance to be tested, and 30 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 35 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 40 having amino acid sequences as depicted in SEQ ID NO: 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 45 sequences such as SEQ ID NO: 2 or SEQ ID NO: 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 5 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. 10 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 NO: 2 or SEQ ID NO: 4. GABAB [sic] receptor-ligand binding is used for the detection in this case. 15 The protein activity of the proteins having the sequences SEQ ID NO: 2 or SEQ ID NO: 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 20 sequences SEQ ID NO: 2 or SEQ ID NO: 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 25 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 30 which regulate the transcription of the sequences SEQ ID NO:1 or SEQ ID NO: 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.], 35 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 p-galactosidase or GFP (= green fluorescent protein) and tested in cells or in 40 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 45 manner (see, for example, Oberdick et al., Science, 248, 1990: 223 - 226).

18 The amino acid sequences SEQ ID NO: 2 or SEQ ID NO: 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 5 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, 10 IgD, IgE or IgA, or their subclasses, such as the subclasses of IgG, or mixtures thereof. IgG and its subclasses, such as IgGi, IgG 2 , IgG2a, IgG2b, IgG 3 and IgGm, are preferred. The IgG subtypes IgGi/K or IgG2b/K are particularly preferred. Fragments which may be mentioned are all truncated or modified antibody fragments 15 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') 2 fragments, or single-stranded fragments. Truncated 20 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 25 manipulated untruncated fragments can also advantageously be used. The antibodies or fragments may be used on their own or in mixtures. 30 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 35 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 40 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 45 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 19 vectors such as pBR322, pUC18/19, 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. 5 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. 10 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 15 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 (BLhm, Klebe, Kubinyi, 1996, 20 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 NO: 2 or SEQ ID NO: 4 or the novel GABAB receptor complex consisting of the already described GABAB 25 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. 30 An alternative route for developing active compounds which interact with the novel GABAB receptor consists in rational drug design (B6hm, 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 35 NO: 2 or SEQ ID NO: 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 GABAB receptor. These substances are then synthesized and tested. 40 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 GABAB receptor complex or of the protein depicted in SEQ ID 45 NO: 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, 20 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 5 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. 10 The present nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 3, its functional equivalents, homologs or derivatives, the protein which it encodes (SEQ ID NO: 2 or SEQ ID NO: 4), or the protein heteromer according to the invention, and also reagents derived 15 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, 20 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 25 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 known quantity of 30 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 35 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. 40 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: 45 a) incubating a biological sample with an antibody which is specifically directed against the protein heteromer or 21 against the protein according to the invention, b) detecting the antibody/antigen complex, 5 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. 10 The invention furthermore relates to a process for discovering substances which bind specifically to a protein having an amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 4, which process comprises one or more of the following steps: 15 a) expressing the protein in eukaryotic or prokaryotic cells, b) incubating the protein with the substances to be tested, 20 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 25 acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 4, or to a nucleic acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3 and thereby elicit inhibitory or activating functional effects on GABAergic signal transmission in central nervous neurones. 30 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 35 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 40 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 45 invention or of the GABAB receptor supplemented with SEQ ID NO: 2 or SEQ ID NO: 4 is in short supply. In the first place, the natural or recombinant protein can be administered directly or, 22 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 5 substances can be found, for example, by ascertaining their effect on the transcription elements of the novel GABAB receptor gene. In situations in which the activity of the GABAB receptor 10 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 NO: 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 NO: 2 or SEQ ID NO: 4 or 15 antibodies or antibody fragments against the protein having the sequence SEQ ID NO: 2 or SEQ ID NO: 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 GABAB receptor activity or the activity of the 20 protein having the sequence SEQ ID NO: 2 or SEQ ID NO: 4. Examples The nucleotide sequence having the structure depicted in SEQ ID 25 NO: 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 30 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 35 sequence which was obtained in this way contains the complete region encoding the sequence SEQ ID NO: 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 40 pharmacological diversity of the GABAB receptor, and also to obtain new specific points of attack for pharmacotherapeutic interventions. Analysis of the sequence of the polypeptide encoded by the 45 present cDNA (= SEQ ID NO: 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, 23 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 5 2.0a19-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 GABAB receptor (best score: 10 36% sequence identity over 804 amino acids with GABAB receptor 1B and 36% sequence identity over 744 amino acids with GABAB receptor 1A) 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 15 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 GABAB receptor mediates signal transmission in the CNS, or regulates 20 this transmission. Figure 2 shows a comparison of the sequence of the protein according to the invention having the sequence SEQ ID NO: 2 with those of the known GABAB [sic] receptor proteins (= GBR) 1A and 25 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. 30 The analysis of the distribution of the mRNA from which the cDNA sequence SEQ ID NO: 1 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 35 mRNA can be detected on a very small scale in rat testis (see Figure 1). 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 GABAs receptor and points to the protein depicted in SEQ ID 40 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, 45 New York".

24 Example 1 Two-hybrid search using the GABAB receptor 1 carboxy terminus 5 The cDNA encoding the carboxy terminus of the GABAB receptor 1A (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' 10 (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-GABAB receptor 1) encodes a protein 15 in which the Gal4-DNA-binding domain is fused to the C terminus of the GABAB 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 20 4x10 6 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 25 each of these colonies was amplified from the pGAD vector using the vector-specific primer Gal4AD3' (5'-AAGAGATCCTAGAACTAGTGGATC-3') and T7 (5'-CGTAATACGACTCACTATAGGGCG-3'), and the amplicon was sequenced. The sequence analysis yielded 5 different overlapping fragments 30 (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 35 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 40 Cloning the cDNA for the novel GABAB receptor component SEQ ID NO: 2 A cDNA fragment which was obtained from the two-hybrid search as 45 described in Example 1 (a, nucleotides 2399-3102 in sequence SEQ ID NO: 1) was radioactively labeled with a- 32 P-dCTP using a random-primed labeling kit (Boehringer Mannheim) in accordance 25 with the manufacturer's instructions. The radioactive probe, which had been denatured by heating, was hybridized for 16 hours (42*C, 5xSSC, 50% formamide) on 18 nitrocellulose filters onto each of which 40,000 plaques from a rat hippocampus cDNA library 5 in bacteriophage X had been transferred; the filters were then washed several times, at 60*C, with 0.2xSSC. Out of 30 positive k 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 10 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 15 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 20 Expressing the mRNA for the novel GABAB receptor component in rat tissues A cDNA fragment which was obtained from the two-hybrid search as 25 described in Example 1 (a, nucleotides 2399-3102 in SEQ ID NO: 1) was radioactively labeled with a- 32 P-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 30 (from Strategene) at 68 0 C for one hour with a multiple-tissue Northern blot (10 pg 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, 35 at 60*C, with 0.1XSSC. 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). 40 Example 4 Expressing the mRNA for the novel GABAB receptor component in rat brain 45 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 5 nucleotides 2463-2498 and nucleotides 2538-2573, respectively, in SEQ ID NO: 1) were radioactively labeled with a- 35 S-dATP using terminal deoxynucleotide transferase (Boehringer Mannheim) in accordance with the manufacturer's instructions. These radioactive probes were applied to horizontal rat brain sections 10 of approximately 15 pm thickness and hybridized at 42*C for 16 hours in 4XSSC, 50% formamide. After that, the sections were washed at 55*C for 30 minutes in 1XSSC and then exposed for 8 days. The autoradiograms for the two oligonucleotides gave a consistent picture (see Figure lb). The strongest signal was 15 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. 20 Example 5 Cloning and sequencing the cDNA for the human form of the novel GABAB receptor component SEQ ID NO: 3 (DNA sequence) and SEQ ID NO: 4 (amino acid sequence) 25 cDNA was synthesized from 5 Rg 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 30 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 RCR [sic] reactions. The primer pairs used in this case were as follows: 35 GBls/GB6as GB15s(hs)/GB18as GB17s/GBllas GB17s/GB16as(hs) GB25s/GB4as 40 GB25s/GB23as(hs) GB25sXbaI/GB23as(hs) GB22s(hs)/GB16as(hs) GBis: 5' -CAGATCCGCAACGAGTCACTCCTG-3' 45 GB2as: 5'-CAGGAGTGACTCGTTGCGGATCTG-3' GB3 s: 5' -CAGTTTGACCAGAATATGGCAGC-3' GB4as: 5'-GCTGCCATATTCTGGTCAAACTG-3' 27 GB6as: 5'-GACCTTCACCTCTCTGCTGTCTTG-3' GB1las: 5' -GAAGGAGGGTGGTACGTGTCTGTG-3' GB15s (hs): 5' -CTACGATGGCATCTGGGTCATC-3' GB16as(hs): 5'-GTCCCATTTCCGTTCCTCTTC-3' 5 GB17s: 5'-CTCAACGACAGCAAGTACATC-3' GB18as: 5' -GATGTACTTGCTGTCGTTGAG-3' GB19as(hs): 5'-GCTCTAGACCGTATTTTATTGCATCGTAG-3' GB22s (hs): 5' -GCGAATTCACAAAAAGACAAGACCATCATCCTG-3' GB2 3as (hs): 5' -GCGAATTCAGGATGGTGAGGGCAGAGAGGATG-3' 10 GB25s: 5'-GTGAATTCGCGGCGCGGCATGGCTTC-3' GB25sXbaI: 5'-GTTCTAGACGCGGCGCGGCATGGCTTC-3' GB27as: 5'-CTGGTCCCGGGTCAGGAAGGAGAC-3' The PCR products were sequenced directly using the primers which 15 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 NO: 3, which 20 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 25 also detected, in addition to the A signal, at positions 360 and 2605 in the sequence SEQ ID NO: 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 NO: 4 amino acid sequence; at position 2605, 30 it would lead to a replacement of threonine with alanine at position 869 of the SEQ ID NO:4 sequence. Base pairs 1 to 8 in SEQ ID NO: 3 were stipulated by the primer GB25s, which was deduced from SEQ ID NO: 1. The possibility cannot be ruled out, therefore, that one or more positions in positions 1 to 8 in SEQ 35 ID NO: 3 is/are different from those indicated. Consequently, one or more of amino acids 1 to 3 in SEQ ID NO: 4 may be different from those indicated. The PCR product GB25sXbaI/GB23as(hs) was subjected to a 40 restriction digestion with XbaI and BglII. The PCR product GB22s(hs)/GB16as(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. 45

Claims (34)

1. A protein heteromer which contains at least one GABAB 5 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 10 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 15 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 20 NO: 4 still being conserved.

3. A nucleic acid sequence which encodes a protein as claimed in claim 2. 25

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 30 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 35 GABAB 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 40 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 GABAB receptor protein. 45 drawings 29

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 5 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 10 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. 15

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 20 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. 25

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. 30

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 activity [sic] for a protein heteromer as 35 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 40 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 45 or 2. 30

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. 5

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. 10

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. 15

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 20 the substance to be tested b) detecting the binding of the substance to be tested to the protein. 25

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

22. A process as claimed in claim 20, wherein the binding of 30 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. 35

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 40 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, 45 b) detecting the nucleic acid as claimed in claim 3 by specific hybridization or PCR amplification, 31 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. 5

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: 10 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, 15 c) comparing the quantities of the antibody/antigen complex with a quantity standard.

25. A process for discovering substances which bind specifically 20 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 25 cells, b) incubating the protein with the substances to be tested, c) detecting the binding of a substance to the receptor or 30 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, 35 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 40 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 45 place specifically with amino acid sequence 785-816 in SEQ ID NO: 2 or 786-817 in SEQ ID NO: 4. 32

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 5 by modulation of the activity of metabotropic receptors.

30. The use as claimed in claim 29, wherein peptides or polypeptides are used as substances. 10

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. 15

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. 20

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 25 as claimed in claim 2. 30 35 40 45