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WO2000015786A1 - Complexe recepteur-gaba metabotrope issu du systeme nerveux central - Google Patents

Complexe recepteur-gaba metabotrope issu du systeme nerveux central Download PDF

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Publication number
WO2000015786A1
WO2000015786A1 PCT/EP1999/006742 EP9906742W WO0015786A1 WO 2000015786 A1 WO2000015786 A1 WO 2000015786A1 EP 9906742 W EP9906742 W EP 9906742W WO 0015786 A1 WO0015786 A1 WO 0015786A1
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Prior art keywords
protein
nucleic acid
seq
gaba
proteins
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PCT/EP1999/006742
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German (de)
English (en)
Inventor
Hans-Christian Kornau
Gisela Eisenhardt
Rohini Kuner
Kirsten Hirschfeld
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Basf-Lynx Bioscience Ag
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Priority claimed from DE19841941A external-priority patent/DE19841941A1/de
Priority claimed from DE1998156066 external-priority patent/DE19856066A1/de
Application filed by Basf-Lynx Bioscience Ag filed Critical Basf-Lynx Bioscience Ag
Priority to EP99947323A priority Critical patent/EP1112363A1/fr
Priority to CA002343574A priority patent/CA2343574A1/fr
Priority to JP2000570313A priority patent/JP2002525052A/ja
Priority to AU60817/99A priority patent/AU6081799A/en
Publication of WO2000015786A1 publication Critical patent/WO2000015786A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention also relates to host organisms or transgenic animals containing the nucleic acid sequences or the recombinant nucleic acid constructs and mono- or polyclonal antibodies which are directed against the isolated proteins.
  • the invention relates to a method for finding substances with specific binding affinity for the new GABA receptors according to the invention or the GABA-receptor complexes according to the invention, a method for the qualitative or quantitative detection of the nucleic acid sequences according to the invention or the proteins according to the invention and one Process for finding substances that bind specifically to a GA-BA receptor according to the invention or to a nucleic acid sequence according to the invention.
  • the invention further relates to the use of the nucleic acid sequences and proteins.
  • GABA ⁇ -amino-butyric acid
  • GABA interacts with two receptor types GABA A and GABA B.
  • GABA A receptors Barnard et al., Trends Neurosci., 10, 1987: 502-509
  • GABA A receptors Barnard et al., Trends Neurosci., 10, 1987: 502-509
  • GABA A receptors Barnard et al., Trends Neurosci., 10, 1987: 502-509
  • These heteromeric complexes form anion channels that are opened upon ligand binding.
  • the release of GABA leads to the activation of these channels to a chloride ion influx, an inhibitory postsynaptic current, into the cell.
  • GABA A receptors are the target of a number of drugs such as benzodiazepines, barbiturates and others [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].
  • GABA A receptors that are intracellularly coupled to G proteins. These receptors are coupled to neuronal potassium and calcium channels via these G proteins.
  • GABA ß receptors GABA ß receptors
  • GABA B receptors are distributed across 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 pre- and ⁇ " postsynaptically.
  • the metabototropic GABA receptors control the release of various neurotransmitters such as GABA, L-glutamate, noradrenaline, dopamine, serotonin, substance P, cholecystokinin, Somatosta- tin and other ligands, agonists or antagonists for GABA ß -.
  • receptors that the Au thoroughlyung a specific the neurotransmitter or regulate neuropeptide can be used to imbalances between the various neurotransmitter systems, such as, in neurodegenerative diseases Excitotoxic side effects of neurological diseases and psychiatric illnesses can be compensated for.
  • GABA B receptors are found on the post-synapse, which activate various potassium channels via Gi proteins.
  • GABA ß receptors are involved in changes in synaptic efficiency that underlie learning and memory processes.
  • GABA B - receptor agonists show positive effects in animal models for chronic pain and cocaine addiction.
  • Antagonists have a positive effect in models of "absence epilepsy" (Bettler et al., Curr. Opin. Neurolbiol., 8, 1998: 345-350). Activation of GABA B receptors dampens overexcited neuronal connections.
  • GABA B receptors are therefore suitable molecular targets for the treatment of epilepsy, stroke, dystonia, cognitive loss, acute and chronic pain, spongiform encephalitis, diseases or injuries to the spinal cord, spasticity and other neurological diseases as well as for the treatment of mental illnesses such as Anxiety, depressive illness, schizophrenia, migraines and others. They are also suitable as targets for the therapy of cocaine or alcohol addicts and as a target for new cognitive enhancers.
  • a GABA B receptor agonist, Baclofen (Lioresal) has been used clinically for the treatment of multiple sclerosis, spasticity and for the consequences of spinal cord injuries (Bowery, Annu. Rev. Pharmacol. Toxicol., 33: 109-147).
  • GABA B receptor agonists are also suspected in the peripheral nervous system (PNS), for example in inflammation and respiratory diseases.
  • PNS peripheral nervous system
  • GA3A B receptor active substances for the treatment of asthma, anaphylactic bronchospasm, airway hyperreactivity and pathological cough.
  • Functional disorders or degenerative diseases of the retina such as retinitis pigmentosa, can also be positively influenced with these substances.
  • Gastroin estinaltraks play in the treatment of diseases, especially refl x esophagitis, ⁇ diseases Magencarcinogenese, stomach ulcers and Er, which are characterized by irregular secretion of stomach acid or pepsinogen, but also in diabetes mellitus and other Diseases of the pancreas and diseases of the immune system.
  • affinities of GABA B receptor agonists for this cloned GABA B receptor are approximately 100 times lower than the values measured in the brain (Kaupmann et al., Nature, 386, 239-246, 1997).
  • GABA B receptor antagonists and agonists and their action can be found in FroestI et al., J. Med. Chem. Vol. 38, 1995: 3297-3312 and 3313-3331.
  • GABA B 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 the development of new pharmaceuticals.
  • the protein heteromer according to the invention comprising at least one GABA B receptor protein and at least one protein with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or one of them by substitution, inversion, insertion or deletion of one or more Sequence obtainable amino acid residues, wherein at least one of the essential biological properties of the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4 or the protein heteromer is retained ⁇ "or with the isolated protein containing those in SEQ ID NO: 2 or SEQ ID NO: 4 amino acid sequence shown or a sequence obtainable therefrom by substitution, inversion, insertion or deletion of one or more amino acid residues, wherein at least one of the essential biological properties of the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4 is obtained remains resolved.
  • the protein heteromers according to the invention are to be understood as meaning GABA receptor complexes advantageously metabotropic GABA receptor complexes containing at least one GABA B receptor protein and at least one protein with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.
  • WO97 / 46675 describes suitable GABA B receptor proteins which can advantageously be contained in the protein heteromers.
  • these GABA B receptor proteins have the sequence names 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 from Homo sapiens).
  • These receptors and the document W097 / 46675 are expressly referred to here.
  • the isolated proteins according to the invention are to be understood as proteins which contain an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or a sequence obtainable therefrom by substitution, inversion, insertion or deletion of one or more amino acid residues, at least one more the essential biological properties of the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4 is retained.
  • certain amino acids can be replaced by those with similar physicochemical properties (space filling, basicity, hydrophobicity, etc.).
  • arginine residues are exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues for glutamic acid residues.
  • SEQ ID NO: 2 or SEQ ID NO: 4 modified proteins have at least 60%, preferably at least 70% and particularly preferably at least 90% sequence identity to the sequences SEQ ID NO: 2 or SEQ ID NO: 4 calculated according to the algorithm of "Altschul et al., J. Mol. Biol., 215, 403-410, 1990 ".
  • the essential biological properties of the proteins or protein heteromers invention are the transmembrane or "membranes areas, the amino-terminal region and much of the carboxy-terminal region of the protein to be understood alone or in protein heteromer (see Figure 2). This protein regions enable the specific biological effect
  • the essential biological properties also include the high affinity binding (Kd ⁇ 10nM) of specific synthetic or natural agonists and antagonists to the proteins according to the invention with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, the signal transmission to an intracellular G protein and the interaction with the known GABA B receptors mentioned above.
  • the proteins SEQ ID NO: 2 and 4 according to the invention are negatively coupled to the enzyme adenylate cyclase and therefore, after activation by GABA or baclofen, bring about a reduction in forskolin-stimulated cAMP production. This result proves that the two 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 the G proteins.
  • GIRKs G-protein coupled inwardly rectifying potassium channels
  • the protein heteromers according to the invention are therefore essential components of the signal cascade triggered by GABA, which leads to the activation of GIRKs.
  • the coupling of the GABA B receptors with GIRKs thus depends on the physical interaction in the protein heteromer according to the invention.
  • the domains responsible for the interaction in the protein heteromer were analyzed by 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 that occur centrally in the intracellular C-terminus of the respective protein.
  • These domains relate to sequence regions of 35 or 32 amino acids (amino acids 887-921 in GABAB receptor 1A or amino acids 785-816 in Seq ID NO: 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: 97-120]). The interaction between these domains specifically mediates the formation of the heteromer, but not the formation of possible homodimers or homomultimers.
  • the isolated protein and its functional variants can advantageously be isolated from the brain of mammals such as Homo sapiens or Rattus norvegicus. Homologs from other mammals are also to be understood as functional variants.
  • the invention further relates to nucleic acid sequences which code for the proteins described above, in particular for those having the primary structure shown in SEQ ID NO: 2 or SEQ ID NO: 4.
  • the nucleic acid sequence from Rat tus norvegicus or Homo sapiens is shown in SEQ ID NO: 1 or in SEQ ID NO: 3.
  • nucleotide sequences according to the invention are SEQ ID No: 1 and SEQ ID No: 3 or their functional equivalents such as, for. B. Allele variants available. Allele variants are understood to mean SEQ ID No: 1 or SEQ ID No: 3 variants which have 60 to 100% homology at the amino acid level, preferably 70 to 100%, very particularly preferably 90 to 100%. Allelic variants include in particular those functional variants which can be obtained by deleting, inserting or substituting nucleotides from the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, at least one of the essential biological properties being retained.
  • Homologous or sequence-related nucleic acid sequences can be isolated from all mammalian species, including humans, by conventional methods by homology screening by hybridization with a sample of the nucleic acid sequences according to the invention or parts thereof.
  • Functional equivalents are also to be understood as homologs of SEQ ID NO: 1 or SEQ ID NO: 3, for example their homologues from other mammals, shortened sequences, single-stranded DNA or RNA of the coding and non-coding DNA sequence.
  • Such functional equivalents can be isolated from other vertebrates such as Mammalia, starting from the DNA sequences described in SEQ ID No: 1 or SEQ ID No: 3 or parts of these sequences, for example using conventional hybridization methods or the PCR technique. These DNA sequences hybridize under standard conditions with the sequences according to the invention. For hybridization, short oligonucleotides of the conserved areas, for example from the transmembrane areas or from the amino-terminal area, which can be determined by comparisons with other transmembrane proteins, specifically other GABA receptors, in a manner known to the person skilled in the art, are advantageously used. However, longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization.
  • DNA hybrids are approx. 10 ° C lower than those of DNA: RNA hybrids of the same length.
  • DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 30 ° C. to 45 ° C.
  • the hybridization conditions are advantageously 0.1 ⁇ SSC and temperatures between about 30 ° C. to 55 ° C., preferably between about 45 ° C.
  • homologs of the sequences SEQ ID No: 1 and SEQ ID No: 3 are to be understood as derivatives such as promoter variants.
  • the promoters which are upstream of the specified nucleotide sequences jointly or individually can be changed by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), but without the functionality or effectiveness of the promoters being impaired.
  • the effectiveness of the promoters can be increased by changing their sequence, or completely replaced by more effective promoters, including organisms of other species.
  • Derivatives are also advantageously to be understood as variants whose nucleotide sequence in the range -1 to -1000 before the start codon have been changed such that the gene expression and / or the protein expression is changed, preferably increased. Derivatives are also to be understood as variants that were changed at the 3 'end.
  • nucleic acids SEQ ID No: 1 or SEQ ID No: 3 alone or the nucleic acids SEQ ID No: 1 or SEQ ID No: 3 and a sequence coding for a GABA B receptor protein with at least one function to link a genetic regulatory element to the recombinant nucleic acid constructs according to the invention.
  • 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 linkage can lead to an increase or decrease in gene expression. With the recombinant nucleic acid constructs thus produced, host organisms are subsequently transformed.
  • the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified so that the natural regulation is switched off and the expression of the genes is increased has been.
  • the gene structure can also have a simpler structure, ie no additional regulation signals are inserted in front of the sequences and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that regulation no longer takes place and gene expression is increased.
  • nucleic acid sequences for the sequences SEQ ID No: 1 or SEQ ID No: 3 and / or for the GABA B receptor proteins can be contained in one or more copies in the gene construct, or can be located on separate gene constructs.
  • Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, lpp-lac, laclq, T7, T5, Contain T3, gal, trc, ara, SP6, 1-PR or in the 1-PL promoter, which are advantageously used in gram-negative bacteria.
  • promoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, lpp-lac, laclq, T7, T5, Contain T3, gal, trc, ara, SP6, 1-PR or in the 1-PL promoter, which are advantageously used in gram-negative bacteria.
  • promoters such as amy and SP02
  • yeast promoters such as ADC1, MFa, AC, P-60, CYC1, GAPDH
  • mammalian promoters such as CaM-Kinasell, CMV, Nestin, L7 , BDNF, NF, MBP, NSE, ß-globin, GFAP, GAP43, tyrosine hydroxylase, kainate receptor subunit 1, glutamate receptor subunit B included.
  • regulatory sequences are intended to enable the targeted expression of the nucleic acid sequences and the protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • the regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase it.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • “Enhancers” are understood to mean, for example, DNA sequences which bring about increased expression via an improved interaction between RNA polymerase and DNA.
  • the locus control regions, silencers or respective partial sequences thereof may be mentioned as further regulatory sequences. These sequences can be used advantageously for tissue-specific expression.
  • a preferred embodiment is the linkage of the nucleic acid sequence according to the invention to a promoter, the promoter being located 5 'upstream.
  • Further regulation signals such as 3 'terminators or polyadenylation signals or enhancers can be used functionally in the nucleic acid construct.
  • nucleic acid construct or gene construct also means complete vector constructs. These vector constructs or vectors are used for expression in a suitable host organism.
  • nucleic acids according to the invention and / or the genes for the GABA 3 receptors are inserted into a host-specific vector which enables optimal expression of the genes in the selected host.
  • Vectors are well known to those skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).
  • vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adeovirus, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA. These vectors can be replicated autonomously in the host organism or can be replicated chromosomally. Linear DNA is advantageously used for the integration into Mammalia.
  • nucleic acid sequences according to the invention or of the recombinant nucleic acid construct can advantageously be increased by increasing the number of gene copies and / or by increasing regulatory factors which have a positive influence on gene expression.
  • regulatory elements can preferably be amplified at the transcription level by using stronger transcription signals such as promoters and enhancers.
  • an increase in translation is also possible, for example, by improving the stability of the mRNA or increasing the reading efficiency of this mRNA on the ribosomes.
  • nucleic acid sequences or homologous genes can be incorporated, for example, into a nucleic acid fragment or into a vector which preferably contains the regulatory gene sequences assigned to the respective genes or promoter activity having an analogous effect.
  • those regulatory sequences are used which increase gene expression.
  • nucleic acid sequences according to the invention can be cloned together with the sequences coding for the GABA B receptors into a single vector and then expressed in the desired organism.
  • each of the nucleic acid sequences described and the sequences coding for the GABA B receptors can also be brought into a single vector and these can be brought into the respective organism separately using conventional methods such as transformation, transfection, transduction, electrophoration or particle gun.
  • 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.
  • vector systems or oligonucleotides can be used which extend the nucleic acids or the nucleic acid construct by certain nucleotide sequences and thus code for modified polypeptides which are used for easier purification.
  • tags in the literature, for. B. Hexa-histidine anchors known or epitopes that can be recognized as antigens of various antibodies (Studier et al., Meth. Zymnzymol., 185, 1990: 60 - 89 and Ausubel et al. [Eds.] M 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York).
  • all organisms are suitable as host organisms which enable expression of the nucleic acids according to the invention, their allele variants, their functional equivalents or derivatives or the recombinant nucleic acid construct alone or together with a sequence which codes for GABA B receptor proteins.
  • Host organisms are, for example, bacteria, fungi, yeasts, plant or animal cells.
  • Preferred organisms are bacteria such as Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms such as Saccharomyces cerevisiae, Aspergillus, higher eukaryotic cells from humans or animals, for example COS, Heia, HEK293, Sf9 or CHO cells .
  • the gene product can also be expressed in transgenic organisms such as transgenic animals, for example mice, rats, sheep, cattle or pigs. In principle, transgenic plants are also conceivable. The transgenic organisms can also be so-called knock-out animals.
  • 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 alone or in combination with a functional or non-functional sequence which codes for GABA B receptor proteins.
  • transgenic animals in their germ cells or in all or part of the somatic cells; or in its germ cells and all or part of the somatic cells, the nucleotide sequence according to the invention was changed by genetic engineering methods or was interrupted by inserting DNA elements.
  • the combination of the host organism and the vectors suitable for the organisms such as plasmids, viruses or phages such as, for example, plasmids with the RNA polymerase / promoter system, which form phages 1, Mu or other tempered phages or transposons and / or further advantageous regulatory sequences ⁇ x- pression system.
  • the term expression systems is preferably understood to mean, for example, the combination of mammalian cells such as CHO cells and vectors such as pcDNA3neo vector or HEK293 cells and CMV vector which are suitable for mammalian cells.
  • the in situ hybridization with the sequence SEQ ID NO: 2 or parts thereof gave a strong expression in the hippocampus, cortex, cerebeilum and in thalamic nuclei (see FIG. 1 and examples).
  • Figures la and lb show the expression analysis of the mRNA corresponding to SEQ ID NO: 1, la shows the Northern blot, lb shows the in situ hybridization (see Examples 3 and 4).
  • the expression pattern overlaps that of the GABA B receptor and indicates an important central nervous function of the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4.
  • the hippocampus is the crucial brain structure for storing new memory content.
  • a protein with the sequence SEQ ID NO: 2 is therefore an interesting target for understanding with regard to learning and memory and for the development of new cognitive enhancers.
  • the hippocampus also affects moods and feelings. Directed against SEQ ID NO: 2 or SEQ ID NO: 4 and their functional equivalents, homologues or derivatives Phar aka thus represent potential antidepressants or anxiolytics and can be used in cognitive disorders. Finally, the hippocampus is heavily involved in temporal lobe epilepsy, which makes a protein with SEQ ID NO: 2 or SEQ ID NO: 4 an attractive target for new drugs against this common disease. Regions are located in the cortex that integrate, process and convert sensory information into suitable reactions. These sensory and motor centers are often the starting points for epileptic seizures.
  • Targeted influencing of the proteins according to the invention or of the protein heteromer according to the invention could reduce the likelihood of convulsions in epilepsy patients.
  • the thalamic nuclei precede the cortex, integrate the senses received through the sensory organs and pass them on to cortical structures. They are often the starting point for generalized seizures.
  • the strong expression of the proteins according to the invention or of the protein heteromer in the thalamic nuclei indicates that its activation or inhibition can contribute to the relief of seizures in epilepsy patients.
  • the cerebellar connections are largely responsible for the fine coordination of the movements. Ataxias and other motor diseases could be based on deregulation of a protein with the nucleic acid sequence according to the invention.
  • the protein heteromers or proteins according to the invention thus represent interesting targets for the development of new substances which are used in the manufacture of medicaments for the treatment of diseases such as neurological diseases such as epilepsy, stroke, mental diseases such as anxiety, manic-depressive diseases, migraines, cognitive losses and other neurological diseases.
  • diseases such as neurological diseases such as epilepsy, stroke, mental diseases such as anxiety, manic-depressive diseases, migraines, cognitive losses and other neurological diseases.
  • the gene for the already known GABA B receptor is located near the chromosomial locus, which is associated with juvenile, myoclonal epilepsy. This correlation could which enable a new diagnostic procedure for this widespread form of epiiepsy.
  • the nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 3 can be used to generate genes for mRNAs which code for these nucleic acids or their functional equivalents, homologs or derivatives, in the murine and in the human genome using conventional methods by homology screening isolate and map and correlate with markers for human hereditary diseases. This enables the gene to be identified as the cause of certain inherited diseases, which considerably simplifies their diagnosis and enables new therapeutic approaches. With the help of nucleic acids as markers, hereditary diseases can be diagnosed.
  • the invention also relates to the use of the nucleic acids according to the invention or parts thereof for gene therapy. Sequences complementary to the nucleic acid according to the invention or parts thereof can also be used for gene therapy.
  • a further possibility of using the nucleotide sequence or parts thereof is the generation of transgenic or knock-out or conditional or region-specific knock-out animals or specific mutations in genetically modified animals (Ausubel et al. [Eds]. 1998, Current Protocols in Molecular Biology, John Wiley & Sons, New York and Torres et al., [Eds.] M 1997, Laboratory protocols for conditional gene targeting, Oxford University Press, Oxford).
  • animal models can be generated which contain valuable further information about the (patho-) physiology of the sequences according to the invention alone or in complex with the GABA B receptor. Animal models produced in this way can represent essential test systems for evaluating novel therapeutic agents that influence the signal transduction of GABA B receptors.
  • the nucleic acid construct, a protein heteromer according to the invention or the protein proteins can be identified which have binding affinities specific to the protein heteromer or the protein, or for identification of nucleic acids which code for proteins which have binding affinities specific to the protein heteromer or the protein .
  • the two-hybrid system or other biochemical methods are advantageously used alone or in combination. Interaction domains of metabotropic receptors and thus pharmacotherapeutic intervention points can be determined in this way.
  • the invention therefore relates to the use of the two-hybrid system or biochemical methods for identifying the interaction domains of metabotropic receptors and the use for pharmacotherapeutic intervention.
  • Structural analyzes of the protein heteromer or the protein according to the invention can be used to specifically find substances which have a specific binding affinity.
  • sequences SEQ ID NO: 1 and SEQ ID NO: 3 described enable, with the aid of the two-hybrid system or other assays, to narrow down the amino acids responsible for the interaction and to find substances with which the interaction between the two metabotropic receptors is influenced can.
  • the use of substances that influence the physical interaction of two metabotropic receptors for the treatment of diseases represents a new pharmacological principle, regardless of the special case described here for the GABA B receptor.
  • the invention further relates to substances, in particular GABA ⁇ receptor antagonists, which specifically reduce or prevent the natural interaction of the GABA B protein with the protein according to SEQ LD NO: 2 or 4.
  • substances in particular GABA ⁇ receptor antagonists, which specifically reduce or prevent the natural interaction of the GABA B protein with the protein according to SEQ LD NO: 2 or 4.
  • Such substances preferably bind to the following sequence areas:
  • these polypeptides themselves and parts of these polypeptides are also suitable as substances which disrupt or prevent the interaction, in particular polypeptides which have a sequence of at least 5 amino acids from one of these sequences (i), (ii) and (iii) have.
  • Another object of the invention is a method for finding substances with specific binding affinity for the protein heteromer or protein according to the invention, which comprises the following steps.
  • Binding is detected by measuring the antagonization or agonization of the GABA B receptor activity or by measuring a physiological effect, such as a change in the calcium, cAMP, IP3 concentration or the membrane potential.
  • Further embodiments of the invention are a method for finding substances which inhibit or intensify the interaction of proteins with amino acid sequences, as shown in SEQ ID NO: 2 or SEQ ID NO: 4, with other metabotropic receptors; a method for finding substances which inhibit or intensify the interaction of ligands with the protein heteromer according to the invention or the proteins according to the invention with amino acid sequences such as SEQ ID NO: 2 or SEQ ID NO: 4 or a method for finding substances which interact with Inhibit or amplify proteins with amino acid sequences such as SEQ ID NO: 2 or SEQ ID NO: 4 with G proteins or other signal transduction molecules.
  • the interaction of proteins with the amino acids according to the invention can be detected with the aid of the two-hybrid system.
  • the methods can also be expressed by expressing the proteins in eukaryotic cells and linking them to a reporter assay for the activation of the GABA B receptor carry out. For example, the change in the cAMP level or the membrane potential is detected.
  • the invention further relates to a method for the qualitative and quantitative determination of proteins with amino acid sequences such as SEQ ID NO: 2 or SEQ ID NO: 4 using specific agonists or antagonists.
  • the GABAB receptor ligand binding is used for detection. -
  • the protein activity of the proteins with the sequences SEQ ID NO: 2 or SEQ ID NO: 4 can be determined via antibodies.
  • the invention therefore furthermore relates to a method for quantifying the protein activity of a protein with the 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 partial sequences thereof, can be used for the tissue-specific expression of this and other genes. This results in the possibility of carrying out brain-specific gene expression of nucleic acid constructs.
  • the start of transcription is then identified on the isolated DNA fragment.
  • the reporter gene can then be linked to other cDNAs in order to create animal models in which the respective cDNA is expressed region-specifically (see, for example, Oberdick et al., Science, 248, 1990: 223-226).
  • Antibodies include both polyclonal, monoclonal, human or humanized or recombinant antibodies or fragments thereof, single chain antibodies or synthetic antibodies.
  • Antibodies according to the invention or their fragments are in principle to be understood as all immunoglobulin classes such as IgM, IgG, IgD, IgE, IgA or their subclasses such as the subclasses of the IgG or their mixtures.
  • IgG and its subclasses such as IgGi, IgG 2 , IgG 2a / ⁇ G 2b ⁇ IgG 3 or IgG M are preferred.
  • the IgG subtypes lgG ⁇ / ⁇ or IgG 2b ⁇ are particularly preferred.
  • Fragments include all shortened or modified antibody fragments with one or two binding sites complementary to the antigen, such as antibody parts with a binding site formed by light and heavy chains corresponding to the antibody, such as Fv, Fab or F (ab ') 2 fragments or single-strand fragments. Shortened double-strand fragments such as Fv, Fab or F (ab ') 2 are preferred.
  • fragments can be obtained, for example, enzymatically by cleaving off the Fc part of the antibodies with enzymes such as papain or pepsin, by chemical oxidation or by genetic engineering manipulation of the antibody genes. Genetically manipulated, unabridged fragments can also be used advantageously.
  • the antibodies or fragments can be used alone or in mixtures.
  • the antibody genes for the genetic engineering manipulations can be isolated, for example, from the hybrid cells (Harlow, E. and Lane, D. 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY; Ausubel et al., [ eds], 1998, Current Protocols in Molecular Bioiogy, John Wiley & Sons, New York).
  • antibody-producing cells are attracted and the mRNA is isolated from the cells in a known manner if the optical density of the cells is sufficient by cell lysis with guanidinium thiocyanate, acidification with sodium acetate, extraction with phenol, chloroform / isoamyial alcohol, precipitation with isopropanol and washing with ethanol.
  • the reverse transcriptase is then used to synthesize cDNA from the mRNA.
  • the synthesized cDNA can be inserted directly or after genetic manipulation, for example by "site directed mutagenesis", introduction of insertions, inversions, deletions or base exchanges into suitable animal, fungal, bacterial or viral vectors and expressed in the corresponding host organisms.
  • Bacterial or yeast vectors such as pBR322, PUC18 / 19, pACYC184, lambda or yeast mu vectors are preferred for cloning the genes and for 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 both as diagnostic reagents and as therapeutic agents for neurological or psychiatric clinical pictures.
  • the cDNA, the genomic DNA, the regulatory elements of the nucleic acid sequences according to the invention, and also the polypeptide, as well as partial fragments thereof can be used in recombinant or non-recombinant form to develop a test system.
  • This test system is suitable for measuring the activity of the promoter or the protein in the presence of the test substance.
  • These are preferably simple measurement methods (colorimetric, luminometric, based on fluorescence or radioactive) which allow the rapid measurement of a large number of test substances (Böhm, Klebe, Kubinyi, 1996, active ingredient design, Spektrum-Verlag, Heidelberg).
  • test systems described allow chemical libraries to be searched for substances which have agonistic or antagonistic effects on SEQ ID NO: 2 or SEQ ID NO: 4 or the new GABA B receptor complex, consisting of the GABA B receptor already described and the protein described in SEQ ID NO: 2 or SEQ ID NO: 4.
  • the identification of such substances is the first step on the way to identifying new types of drugs that have a specific effect on GABAergic signal transduction.
  • the determination of the amount, activity and distribution of the new GABA B receptor complex or of the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4 or its underlying mRNA in the human body can be used for diagnosis, predisposition detection and monitoring serve for certain diseases.
  • the sequence of the cDNA of the sequences SEQ ID NO: 2 or SEQ ID NO: 4 and the genomic DNA can be used to make statements about the genetic causes and predispositions of certain diseases. You can use both DNA / RNA samples as well Various types of antibodies can be used.
  • the nucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 3 or parts thereof described in the form of suitable samples serves to detect point mutations or deletions / insertions / rearrangements.
  • Tested reagents oligonucleotides, antibodies, peptides
  • the invention further relates to a method for the qualitative and quantitative detection of a nucleic acid according to the invention in a biological sample, which comprises the following steps:
  • the invention also relates to a method for the qualitative and quantitative detection of a protein heteromer according to the invention or a protein according to the invention in a biological sample, which comprises the following steps:
  • a biological sample is usually taken from a healthy organism.
  • the invention further relates to a method for finding substances which bind specifically to a protein with an amino acid sequence - SEQ ID NO: 2 or SEQ ID NO: 4, which comprises one or more of the following steps:
  • the invention relates to a method for finding substances that are specific to a protein with 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 NO: 1 or SEQ ID NO: 3 bind and thereby cause inhibitory or activating functional effects on the GABA mountains signal transmission in central nervous neurons.
  • GABA neurotransmitter system
  • glutamate glutamate
  • GABA B receptor activity can lead to an imbalance between the neurotransmitter systems and often to neuronal overexcitation, which can cause a variety characterized neurological diseases such as epilepsy, stroke and its consequences and others.
  • a lack of neuronal activity, which characterizes dementia, for example, can result if presynaptic GABA B receptors on glutamatergenic synapses are overactivated and inhibit the transmitter release so strongly that stimulus transmission via the synapse is no longer possible.
  • the nucleotide sequence with the structure shown in SEQ ID NO: 1 was identified from a rat brain cDNA library. When looking for proteins that interact with the intracellular carboxy terminus of the known GABA B receptor, the nucleotide sequence was found. In the experiment, a rat brain cDNA library with the two-hybrid system was found for interaction partners of the carboxy terminus described above
  • GABA B receptor searched. Several overlapping fragments of an unknown cDNA were found. Using these fragments, a 5 kb fragment of the unknown cDNA was isolated from a cDNA library from the rat hippocampus by homology screening and then sequenced. The cDNA sequence obtained in this way contains the complete coding region for the sequence SEQ ID NO: 2.
  • the discovery and molecular characterization of interaction partners of the known cloned GABA B receptor enables the physiological properties and the biochemical and pharmacological diversity of the GABA B receptor can be better understood, and new concrete targets for pharmacotherapeutic interventions can be obtained.
  • sequence analysis of the polypeptide encoded by the present cDNA suggests that it is a metabotropic receptor. It contains an amino-terminal signal sequence [amino acids 1-40 (G. von Heijne, NAR, 14, 4683, 1986)] and seven characteristically arranged hydrophobic areas, which are highly likely to span the plasma membrane. The presence of these seven hydrophobic areas is a characteristic of metabotropic receptors. Sequence comparisons (Altschul et al., J. Mol.
  • GBR GABAB receptor proteins
  • the analysis of the distribution of the mRNA from which the cDNA sequence SEQ ID NO: 1 was derived was carried out by Northern blot and by in-situ hybridization on rat brain sections. Analysis in 10 different rat tissues revealed brain-specific expression of a 5-6 kb mRNA. A smaller mRNA can be detected in rat testis to a very small extent (see FIG. 1). In situ hybridization resulted in strong expression in the hippocampus, cortex, cerebellum and thalamic nuclei. The expression pattern overlaps that of the GABA B receptor and indicates an important central nervous function of the protein shown in SEQ ID NO: 2.
  • the cDNA coding for the carboxy terminus of the GABA B receptor 1A was primed with the specific primers GABA-CT5 * (5 v -GCGAATTCCGCAGGCTGAT-CACCCGAGGG-3 N ) and GABA -CT3 v (5 v -GCAGTCGACTCACTTGTAAAGCAAATG- TACTCG-3 v ) from rat brain cDNA in a polymerase chain reac-
  • the pGAD plasmid DNA was purified from two different positive colonies (a and e) and co-transformed into the yeast strain HF7c with different pGBT constructs. Activation of the reporter genes His3 and lacZ was only found in combination with the construct pBGT-GABA B receptor 1.
  • a cDNA fragment obtained from the two-hybrid search according to example 45 (a, nucleotides 2399-3102 in sequence SEQ ID NO: 1) was labeled with - 32 P using the random primed labeling kit (Boehringer Mannheim) according to the manufacturer's instructions -dCTP radioactive mar- kiert.
  • the radioactive probe denatured by heating, was hybridized for 16 hours on 18 nitrocellulose filters to which 40,000 plaques of a cDNA library from rat hippocampus in the bacteriophage ⁇ had been transferred (42 ° C, 5xSSC, 50% formamide). and then washed several times with 0.2xSSC, 60 ° C.
  • a cDNA fragment obtained from the two-hybrid search according to Example 1 was radioactive with the random primed labeling kit (Boehringer Mannheim) according to the manufacturer's instructions with ⁇ - 32 P-dCTP marked.
  • the radioactive probe, denatured by heating, was isolated with a multiple tissue Northern blot (10 ⁇ g total RNA from rat brain, liver, lung, heart, kidney, testicle, muscle and intestine, according to "Chomczinski and Sacchi, Anal.
  • Two antisense oligonucleotides (reverse complementary to nucleotides 2463-2498 or nucleotides 2538-2573 in the sequence SEQ ID NO: 1) were radioactively labeled with ⁇ - 35 S-dATP using terminaler deoxynucleotide transferase (Boehringer Mannheim) according to the manufacturer's instructions . These radioactive probes were applied to horizontal rat brain sections about 15 ⁇ m thick and hybridized in 4xSSC, 50% formamide at 42 ° C for 16 hours. The sections were then washed in lxSSC, 55 ° C. for 30 minutes and exposed for 8 days. The autoradiograms for both oligonucleotides showed an identical picture (see FIG. 1 b). The strongest signal was found in Purkinje cells of the cerebellum, likewise strong signals in the cortex, hippocampus and in various thalamic nuclei as well as weaker signals in the cerebellar granule cells.
  • cSNA was synthesized using Superscript Reverser Transcriptase (Gibco BRL) according to the manufacturer's instructions.
  • Gibco BRL Superscript Reverser Transcriptase
  • GBls 5 '-CAGATCCGCAACGAGTCACTCCTG-3' GB2as: 5 '-CAGGAGTGACTCGTTGCGGATCTG-3' GB3S: 5 '-CAGTTTGACCAGAATATGGCAGC-3' GB4as: 5 '-GCTGCCATATTCTGGTCTCCTACTGAC' 5 GAAGGAGGGTGGTACGTGTCTGTG-3 'GB15s (hs): 5' -CTACGATGGCATCTGGGTCATC-3 'GB16as (hs): 5' -GTCCCATTTCCGTTCCTCTTC-3 'GB17s: 5' -CTCAACGACAGCAAGTCATGGT3 'GBGBAT18GAS-3' : 5 '-GCTCTAGACCGTATTTTATTGCATCGTAC-3' GB22S (hs): 5 '-GCGAATTCACAAAAAGACAAGACCATCATCCTG-3' GB23as (hs): 5
  • the PCR products were sequenced directly with the primers that had already been used for the amplification or with the primers mentioned above. New primers were derived from the sequences obtained and used for the PCR reactions and sequencing (list of primers see above).
  • the PCR product GB25sXbaI / GB23as (hs) was subjected to a restriction digest with Xbal and Bglll.
  • the PCR product GB22s (hs) / GBl ⁇ as (hs) was subjected to a restriction digest with Bglll and Xhol. educated.
  • the two PCR products were cloned into a pBSIIKS (-) vector (Stratagene) pre-cut with Xbal and Xhol (pBS-hsGB).

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Abstract

L'invention concerne des protéines isolées codant les récepteurs-GABA métabotropes et formant conjointement avec des protéines à récepteur GABAB, un complexe récepteur-GABA métabotrope (= hétéromère de protéine), ainsi que des séquences d'acide nucléique ou des produits de recombinaison d'acide nucléique recombinés qui codent les protéines. L'invention concerne en outre des organismes hôtes ou des animaux transgéniques contenant lesdites séquences d'acide nucléique ou lesdits produits de recombinaison d'acide nucléique recombinés, ainsi que les anticorps mono- ou polyclonaux dirigés contre les protéines isolées. L'invention concerne de plus un procédé permettant de détecter des substances à affinité spécifique de liaison sur les nouveaux récepteurs-GABA ou les nouveaux complexes de récepteurs-GABA obtenus selon l'invention, un procédé permettant la mise en évidence qualitative ou quantitative des séquences d'acide nucléique obtenues selon l'invention ou des protéines obtenues selon l'invention, ainsi qu'un procédé permettant de détecter des substances se liant de manière spécifique sur un récepteur-GABA ou une séquence d'acide nucléique selon l'invention. L'invention concerne par ailleurs l'utilisation des séquences d'acide nucléique et des protéines. L'invention concerne pour finir des substances permettant d'influer sur l'activité de récepteurs-GABA métabotropes par action sur la stabilité de l'hétéromère de protéine.
PCT/EP1999/006742 1998-09-14 1999-09-11 Complexe recepteur-gaba metabotrope issu du systeme nerveux central WO2000015786A1 (fr)

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EP99947323A EP1112363A1 (fr) 1998-09-14 1999-09-11 Complexe recepteur-gaba metabotrope issu du systeme nerveux central
CA002343574A CA2343574A1 (fr) 1998-09-14 1999-09-11 Complexe recepteur-gaba metabotrope issu du systeme nerveux central
JP2000570313A JP2002525052A (ja) 1998-09-14 1999-09-11 中枢神経系由来の代謝共役型ｇａｂａ受容体複合体
AU60817/99A AU6081799A (en) 1998-09-14 1999-09-11 Metabotropic gaba receptor complex issued from the central nervous system

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DE19841941A DE19841941A1 (de) 1998-09-14 1998-09-14 Neuer metabotroper-Rezeptor-Komplex aus dem zentralen Nervensystem
DE19841941.4 1998-09-14
DE19856066.4 1998-12-04
DE1998156066 DE19856066A1 (de) 1998-12-04 1998-12-04 Neuer metabotroper GABA-Rezeptor-Komplex aus dem zentralen Nervensystem

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Citations (4)

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WO1999020751A1 (fr) * 1997-10-17 1999-04-29 Synaptic Pharmaceutical Corporation Adn codant un polypeptide gababr2 et ses utilisations
WO1999040114A1 (fr) * 1998-02-05 1999-08-12 Merck & Co., Inc. Nouvelles sequences d'adn codant le recepteur gaba¿b?
WO1999051636A2 (fr) * 1998-04-03 1999-10-14 Nps Pharmaceuticals, Inc. Recepteur gaba b
WO1999051641A1 (fr) * 1998-04-03 1999-10-14 Nps Pharmaceuticals, Inc. Recepteurs d'hybridation de proteine g et recepteurs gabab chimeres

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Publication number Priority date Publication date Assignee Title
WO1999020751A1 (fr) * 1997-10-17 1999-04-29 Synaptic Pharmaceutical Corporation Adn codant un polypeptide gababr2 et ses utilisations
WO1999040114A1 (fr) * 1998-02-05 1999-08-12 Merck & Co., Inc. Nouvelles sequences d'adn codant le recepteur gaba¿b?
WO1999051636A2 (fr) * 1998-04-03 1999-10-14 Nps Pharmaceuticals, Inc. Recepteur gaba b
WO1999051641A1 (fr) * 1998-04-03 1999-10-14 Nps Pharmaceuticals, Inc. Recepteurs d'hybridation de proteine g et recepteurs gabab chimeres

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Title
JONES K. A. ET AL.: "GABAB receptors function as heteromeric assembly of the subunits GABABR1 and GABABR2.", NATURE, vol. 396, 17 December 1998 (1998-12-17), pages 674 - 679, XP002116148, ISSN: 0028-0836 *
KAUPMANN K. ET AL.: "GABAB-RECEPTOR SUBTYPES ASSEMBLE INTO FUNCTIONAL HETEROMERIC COMPLEXES", NATURE, vol. 396, no. 17, December 1998 (1998-12-01), pages 683 - 687, XP000867007, ISSN: 0028-0836 *
KUNER R. ET AL.: "Role of heteromer formation in GABA-B receptor function.", SCIENCE, vol. 283, 1 January 1999 (1999-01-01), pages 74 - 77, XP002131319 *
WHITE J. H. ET AL.: "Heterodimerization is required for the formation of a functional GABAB receptor", NATURE, vol. 396, 17 December 1998 (1998-12-17), pages 679 - 682, XP002116149, ISSN: 0028-0836 *

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