WO1999051636A2 - Gaba b receptor - Google Patents

Abstract

The present invention features a novel GABAB receptor subtype ('GABABR2'). The cDNA sequence encoding GABABR2 is shown in Figures (1a-1n) as SEQ. ID. NO: 1. The GABABR2 amino acid sequence is provided in Figures (2a-2f) as SEQ. ID NO: 4.

Description

GABA_B RECEPTOR

RELATED APPLICATIONS The present application claims priority to Garrett et' al . U.S. Serial No. 60/080,676, filed April 3, 1998, which is hereby incorporated by reference herein in its entirety including the drawings.

FIELD OF THE INVENTION The present invention relates to a GABA_B receptor, nucleic acid encoding a GABA_B receptor, and uses of a GABA_B receptor and nucleic acid encoding a GABA_B receptor .

BACKGROUND The references cited herein are not admitted to be prior art to the claimed invention. GABA_B receptors are metabotropic receptors coupled to guanine-nucleotide-binding proteins (G-proteins) . GABA_B receptors modulate synaptic transmission by inhibiting presynaptic transmitter release and by increasing K⁺ conductance responsible for long-lasting inhibitory postsynaptic potentials. (Kaupmann et al . , Nature 386: 239-246 , 1997, hereby incorporated by reference herein.)

GABA_B receptors are found in the mammalian brain, in locations outside of the brain, and in lower species. Outside of the brain, GABA_B receptors have been identified on axon terminals and ganglion cell bodies of the autonomic nervous system, on fallopian tube and uterine intestinal smooth muscle cells, in the kidney cortex, urinary bladder muscle and on testicular interstitial cells. ( See, Bowery, Annu . Rev. Pharmacol . Toxicol . 33:109-147, 1993, hereby incorporated by reference herein.)

GABA_B receptors have been targeted to achieve therapeutic effects. Kerr and Ong, DDT 1:371-380, 1996, describe different compounds indicated to be GABA_B receptor agonists and GABA_B receptor antagonists. Kerr and Ong also review therapeutic implications of affecting GABA receptor activity including, spasticity and motor control, analgesia, epilepsy, cognitive effects, psychiatric disorders, alcohol dependence and withdrawal, feeding behavior, cardiovascular and respiratory functions, and peripheral functions. Bittiger et al . , Tips 4:391-394, 1993, review therapeutic applications of GABA_B receptor antagonists. Potential therapeutic applications noted by Bittiger et al . include cognitive processes, epilepsy, and depression.

Kaup ann et al . , Nature 386: 239-246, 1997, indicate that they cloned GABA_B receptors. Two GABA_B receptor proteins were indicated to be cloned from rat brain: GABA_BRla and GABA_BRlb. GABA_BRla differs from GABA_BRlb in that the N-terminal 147 residues are replaced by 18 amino acids. GABA_BRla and GABA_BRlb appear to be splice variants. The cloned GABA_B receptors were indicated to negatively couple to adenylyl cyclases and show sequence similarity to the metabotropic receptors for L- glutamate (mGluR) .

Kaupmann et al . , Nature 386: 239-246, 1997, indicate that bestfit sequence alignments with GABA_B and different mGluR subtypes indicates 18-23% amino acid sequence identity and 43- 48% related residues. (Devereux et al . , Nucleic Acids Res . 12:387-395, 1984, was referenced for carrying out bestfit sequence alignments.) No significant sequence similarity was found with GABA_A or GABA_C receptors, or with other G-protein- coupled receptors which were not mGluR.

Kaupmann et al . , International Application Number PCT/EP97/01370, International Publication Number WO 97/46675, indicate that they have obtained rat GABA_B clones, GABA_BRla and GABA_BRlb; and human GABA_B clones, GABA_BRla/b (representing a partial receptor clone) and GABA_BRlb (representing a full- length receptor clone) . Amino acid sequence information, and encoding cDNA sequence information, is provided for the different human GABA_B clones.

SUMMARY OF THE INVENTION

The present invention features a novel GABA_B receptor subtype ("GABA_BR2") . The cDNA sequence encoding GABA_BR2 is shown in Figures la-ln as SEQ. ID. NO. 1. The GABA_BR2 amino acid sequence is provided in Figures 2a-2f as SEQ. ID. NO. 4. Thus, a first aspect of the present invention describes a purified nucleic acid containing at least 18 contiguous nucleotides of SEQ. ID. NO. 1 which provides the nucleic acid encoding GABA_BR2. Preferably, the nucleic acid contains at least 27 contiguous nucleic acids, more preferably at least 45 contiguous nucleic acids, or most preferably the entire nucleic acid sequence provided in SEQ. ID. NO. 1. Advantages of longer- length nucleic acid include producing longer-length protein fragments having the sequence of GABA_BR2 which can be used, for example, to produce antibodies; and increased nucleic acid probe specificity under higher stringent hybridization assay conditions .

By "purified" in reference to nucleic acid is meant the nucleic acid is present in a form (i.e., its association with other molecules) other than found in nature. For example, a purified receptor nucleic acid is separated from one or more nucleic acids which are present on the same chromosome. Preferably, the purified nucleic acid has been separated from at least 90% of the other nucleic acids present on the same chromosome. More preferably, the nucleic acid has been substantially purified such that it represents at least 75%, more preferably at least 85%, and most preferably at least 95% of the total nucleic acids present.

Another example of purified nucleic acid is recombinant nucleic acid. Preferably, recombinant nucleic acid contains nucleic acid encoding GABA_BR2 or GABA_BR2 fragments cloned in a vector. The vector contains the necessary elements for introducing heterologous nucleic acid into cells for either expression or replication. Preferably, the vector is an expression vector containing elements needed for expressing a cloned nucleic acid sequence to produce a polypeptide. The expression vector contains a promoter region directing the initiation of RNA transcription, and DNA sequences which when transcribed into RNA signal protein synthesis initiation.

Recombinant nucleic acid may contain nucleic acid encoding for GABA_BR2, a GABA_BR2 fragment, or a GABA_BR2 derivative, under the control of genomic GABA_BR2 nucleic acid regulatory elements, or under the control of exogenous regulatory elements including an exogenous promoter. By "exogenous" is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for GABA_BR2.

Another aspect of the present invention features a purified nucleic acid encoding at least 6 contiguous amino acids of the GABA_BR2 amino acid sequence which is provided as SEQ. ID. NO. 4. Due to the degeneracy of the genetic code, different combinations of nucleotides encode for the same polypeptide. Thus, numerous GABA_BR2 and GABA_BR2 fragments having the same amino acid sequences can be encoded for by different nucleic acid sequences. In preferred embodiments, the nucleic acid encodes at least 12, at least 18, at least 54 contiguous amino acids, or the entire amino acid sequence provided in SEQ. ID. NO. 4.

Another aspect of the present invention features a recombinant cell. The recombinant cell, which can be a tissue cell, is made up of a recombinant nucleic acid encoding GABA_BR2, a functional GABA_BR2 derivative, or a fragment thereof, and a cell able to express the nucleic acid. Recombinant cells have various uses including acting as biological factories to produce large amounts of polypeptides encoded for by the recombinant nucleic acid, as tools for screening for compounds which modulate GABA_BR activity, and as research tools to study the effects of GABA_BR activity.

Another aspect of the present invention features a purified nucleic acid comprising a nucleic acid sequence region substantially complementary to a sequence region of the SEQ. ID. NO. 1 or the perfect complement of SEQ. ID. NO. 1. Such nucleic acid can be used, for example, to specifically detect the presence of nucleic acid encoding for GABA_BR2 or a close relative thereof.

Substantially complementary nucleic acid regions contain at least 18 nucleotides in a stretch of 20 contiguous nucleotides which are complementary. Complementary nucleic acid form Watson- Crick A-T, G-C, and A-U, hydrogen bonds. More preferably, the nucleic acid comprises a nucleotide sequence of 20 contiguous nucleotides which has at least 19 bases, most preferably 20 bases, complementary to the nucleic acid sequence provided in SEQ. ID. NO. 1 or the perfect complement of SEQ. ID. NO. 1.

Another aspect of the present invention features a purified polypeptide having at least 6 contiguous amino acids of the GABA_BR2 amino acid sequence. By "purified" in reference to a polypeptide is meant that the polypeptide is in a form (i.e., its association with other molecules) distinct from naturally occurring polypeptides . Preferably, the polypeptide has been substantially purified to represent at least 75%, more preferably 85%, most preferably 95% of the total protein present in a preparation. In preferred embodiments, the purified polypeptide has at least 12 contiguous, at least 18 contiguous, at least 54 contiguous, or the entire amino acid sequence of SEQ. ID. NO. 4.

Another aspect of the present invention features a GABA_BR2- binding agent comprising a molecule which binds to a polypeptide consisting of the amino acid sequence of SEQ. ID. NO. 4. The binding agent is preferably a purified antibody. Other examples of binding agents include organic compounds which bind to GABA_BR2. By "purified" in reference to a binding agent, such as an antibody, is meant that the binding agent is in a form (i.e., its association with other molecules) distinct from a naturally occurring binding agent, if the binding agent is found in nature. Preferably, the binding agent is an antibody provided as a purified preparation representing at least 1%, more preferably at least 50%, more preferably at least 85%, most preferably at least 95% of the total protein in the preparation.

Another aspect of the present invention describes a method of making a GABA_BR2 or a fragment thereof. The method is carried out by incubating recombinant cells containing nucleic acid encoding GABA_BR2 or a fragment thereof under conditions where the nucleic acid is expressed.

Another aspect of the present invention describes a method of selecting for compounds able to modulate GABA_BR activity. The method comprises the steps of (a) contacting a recombinant cell functionally expressing GABA_BR2 with a first test compound; and (b) measuring the ability of said test compound to affect GABA_BR activity. Compounds modulating GABA_BR activity either evoke a GABA_BR activity, potentiate GABA_BR activity, or inhibit a GABA_BR activity. Cells functionally expressing GABA_BR2 also express GABA_BRla and/or GABA_BRlb.

Preferably, the ability of a plurality of different test compounds to affect GABA_BR activity are tested. In preferred embodiments at least 5, at least 10, at least 50 different compounds, and at least 100 different compounds are tested over a span of one week.

Other aspects of the present invention describe coexpression systems and the use of such systems to measure the activity at, or screen compounds active at, GABA_BRla, GABA_BRlb, or GABA_BR2, preferably GABA_BR2. The coexpression systems comprise at least one of GABA_BRla and GABA_BRlb, GABA_BR2, and Gqo5.

Other aspects of the present invention describe coexpression systems and the use of such systems to measure the activity at, or screen compounds active at, GABA_BRla, GABA_BRlb, or GABA_BR2.

The coexpression systems comprise at least one of GABA_BRla or

GABA_BRlb, coexpressed with GABA_BR2 and Gqo5. The presence of Gqo5 provides for signal transduction swapping allowing for receptor activity to be measured by mobilization of intracellular calcium mediated by the activation of phospholipase C.

Assays using the coexpression systems described above can be used to screen chemical libraries for compounds that modulate

GABA_B receptors. For example, in different embodiments, a library of compounds containing 10 or more compounds is screened at once; and 10 or more compounds are individually tested over the course of eight hours.

Preferably, the coexpression system is present in an isolated cell. An "isolated cell" includes tissue cells and refers to a cell present in a different environment (including a different concentration), than it is normally found in nature. In other aspects, the invention describes transgenic nonhuman mammals containing a transgene encoding GABA_BR2, a

GABA_BR2 fragment, or a derivative thereof; or a gene affecting the expression of GABA_BR2; and methods of creating a transgenic nonhuman mammal containing a transgene encoding an GABA_BR2, a

GABA_BR2 fragment, or a derivative thereof.

Various examples are described herein. These examples are not intended in any way to limit the claimed invention. Other features and advantages of the invention will be apparent from the following drawing, the description of the invention, the examples, and the claims. BRIEF DESCRIPTION OF DRAWINGS Figures la-ln illustrate the nucleic acid sequences encoding for the human GABA_BR2 designated SEQ. ID. NO. 1, human GABA_BRla designated SEQ. ID. NO. 2, and human GABA_BRlb designated SEQ. ID. NO. 3.

Figures 2a-2f illustrate the amino acid sequences of the human GABA_BR2 (SEQ. ID. NO. 4); the rat GABA_bRla (SEQ. ID. NO. 5); the rat GABA_bRlb protein (SEQ. ID. NO. 6); the human GABA_bRla (SEQ. ID. NO. 7); and the human GABA_bRla (SEQ. ID. NO. 8). Figures 3a-3d provides the human calcium receptor nucleic acid sequence and the encoded for amino acid sequence.

Figure 4 illustrates functional expression of GABA_BR2 in Xenopus oocytes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features GABA_BR2. GABA_BR2 is closely related to GABA_BRla and GABA_BRlb. Nucleic acid encoding for human GABA_BR2 has a sequence similarity of about 50% with nucleic acid encoding rat GABA_BRla and rat GABA_BRlb . Human GABA_BR2 has a sequence identity of about 40% with rat GABA_BRla and GABA_BRlb amino acid sequence.

Nucleic acid encoding GABA_BR2 was cloned by first identifying a human nucleic acid sequence approximately 38% identical to the nucleic acid sequence of rat GABA_BR1. Exact match polymerase chain reaction (PCR) primers were designed based on sequences from the identified sequence and used to amplify human GABA_BR2 nucleic acid from a human cerebral cortex cDNA library. A PCR product encoding human GABA_BR2 was isolated and cloned. Northern blot analysis revealed that an approximately 6.3 Kb human GABA_BR2 transcript was abundantly expressed in the human brain. Expression was not detected in the heart, placenta, lung, liver, skeletal muscle, kidney or pancreas under conditions where GABA_BR2 transcript was identified in the human brain. Within the human brain GABA_BR2 is broadly expressed at variable levels.

Compounds modulating GABA_BR activity can be obtained, for example, by screening a group, or library, of compounds to identify those compounds having the desired activity and then synthesizing such compounds. Thus, included in the present invention is a method of making a GABA_BR active compound by first screening for a compound having desired properties and then chemically synthesizing that compound.

Nucleic Acid Encoding GABA_BR2

Nucleic acids encoding GABA_BR2 have a variety of different uses including one or more of the following: (1) producing receptor proteins which can be used, for example, for structure determination, to assay a molecule's activity on a receptor, and to obtain GABA_BR2 modulatory agents; (2) being sequenced to determine a receptor's nucleotide sequence which can be used, for example, as a basis for comparison with other receptors to determine conserved regions, determine unique nucleotide sequences for normal and altered receptors, and to determine nucleotide sequences to be used as target sites for antisense nucleic acids, ribozymes, hybridization detection probes, or PCR amplification primers; (3) as hybridization detection probes to detect the presence of a native receptor and/or a related receptor in a sample; (4) as PCR primers to generate particular nucleic acid sequence regions, for example, to generate regions to be probed by hybridization detection probes; and (5) to provide an extracellular domain, transmembrane domain, or extracellular domain for use in the construction of a chimeric receptor. Hybridization probes and primers based on the GABA_BR2 sequence information provided herein can be used, for example, to obtain nucleic acid from different sources or to identify the presence of GABA_BR2 nucleic acid in a sample. Nucleic acid encoding proteins related to human GABA_BR2 can be obtained from human and nonhuman sources. Such related nucleic acids are useful for identifying important GABA_BR2 structural motifs and may also provide new therapeutic target sites.

Primer hybridization specificity to target nucleic acid can be adjusted by varying the hybridization conditions. When annealing at higher stringency conditions of 50-60°C, sequences which are greater than about 75% complementarity to the primer will be amplified. By employing lower stringency conditions, annealing at 35-37°C, sequences which are greater than about 40- 50% complementarity to the primer will be amplified. Hybridization assay probes can be designed to detect the presence of a particular nucleic acid target sequence perfectly complementary to the probe and target sequences of lesser complementarity by varying the hybridization conditions and probe design. Factors affecting probe design, such as length, G and C content, possible self-complementarity, and wash conditions, are well known in the art. (See, for example, Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory Press (1989).) Sambrook et al . , Molecular Cloning, also discusses the design and use of degenerative probes based on polypeptide sequence information.

Preferably, the nucleic acid probes targeted to GABA_BR2 nucleic acid distinguish GABA_BR2 nucleic acid from GABA_Bla and GABA_Blb nucleic acid. Such probes are readily designed by comparing the nucleic acid sequences of target GABA_BR2, and non- target GABA_Bla and GABA_Blb, to obtain probes having proper probe: target and probe : non-target T_m characteristics. Preferably, the probe: target duplex T_m is at least about 5°C greater than the probe : non-target T_m. Probes specific for a target contain a target complementary region and may also contain target non-complementary regions. The target non-complementary regions, if present, are designed not to affect the specificity of the probe. An example of a target non-complementary region is a nucleic acid sequence used as a capture sequence in a sandwich assay, where the capture sequence does not hybridize to target or non-target nucleic acids. (See, Stabinsky, U.S. Patent No. 4,739,044, and Ranki et al . , U.S. Patent No. 4,563,419, both of which are incorporated by reference herein.) The probes can be used under conditions of proper stringency conditions where target and non-target nucleic acid are distinguished. As the stringency conditions are increased, the complementarity of two nucleic acids required to form a stable duplex is also increased. As a general guideline, high stringency conditions (e.g., hybridization at 50-65°C, 5X SSPC, 50% formamide, wash at 50-65°C, 0.5X SSPC) can be used to obtain hybridization between nucleic acid sequences having regions which are greater than about 90% complementary. Low stringency conditions (e.g., hybridization at 35-37°C, 5X SSPC, 40-45% formamide, wash at 42°C IX SSPC) can be used so that sequences having regions greater than 35-45% complementarity will hybridize to the probe.

If desired, nucleic acid probes may be labeled with a detectable label using techniques well known in the art.

Examples of detectable labels include radiolabels, enzymes, fluorescent molecules, and chemiluminescent molecules.

Any tissue can be used as a source for genomic DNA. However, with respect to RNA, the most preferred source is tissues which express elevated levels of GABA_BR2 or related proteins .

Specific nucleic acids can also be produced enzymatically using a host transformed with a plasmid encoding for the desired nucleic acid. Additionally, standard techniques for chemically synthesizing nucleic acids include solid phase phosphoramidite chemical synthesis.

GABA_BR2 polypeptides

GABA_BR2 polypeptides made up of GABA_BR2, GABA_BR2 fragments, and derivatives thereof have different uses including, being used to produce antibodies to determine the presence of the protein, and being used to screen for compounds able to bind to the protein. GABA_BR2 polypeptides are preferably produced using recombinant nucleic acid techniques. Polypeptides can also be synthesized using solid phase techniques. Solid-phase synthesis is commenced from the carboxy- terminal end of the peptide using an α-amino protected amino acid. BOC protective groups can be used for all amino groups even though other protective groups are suitable. For example, BOC-lys-OH can be esterified to chloromethylated polystyrene resin supports. The polystyrene resin support is preferably a copolymer of styrene with about 0.5 to 2% divinylbenzene as a cross-linking agent which causes the polystyrene polymer to be completely insoluble in certain organic solvents. See Stewart et al . , Solid-Phase Peptide Synthesis (1969), W.H. Freeman Co., San Francisco; and Merrifield, J. Am . Chem . Soc . 85:2149-2154, 1963. These and other methods of peptide synthesis are also exemplified by U.S. Patent Nos. 3,862,925; 3,842,067; 3,972,859; and 4,105,602. GABA_BR2 derivatives, and nucleic acid encoding for GABA_BR2 derivatives can be produced using techniques well known in the art based upon the present disclosure. GABA_BR2 derivatives have a sequence similarity of at least 70%, more preferably at least 90%, even more preferably at least 95% sequence similarity to the amino acid sequence provided in SEQ. ID. NO. 4. Sequence similarity is preferably determined using BLASTN (Altschul et al . , J. Mol . Biol . 225:403-410, 1990.)

Examples of specific types of derivatives include amino acid alterations such as deletions, substitutions, additions, and amino acid modifications. A "deletion" refers to the absence of one or more amino acid residue (s) in the related polypeptide. An "addition" refers to the presence of one or more amino acid residue (s) in the related polypeptide. Additions and deletions to a polypeptide may be at the amino terminus, the carboxy terminus, and/or internal. Amino acid "modification" refers to the alteration of a naturally occurring amino acid to produce a non-naturally occurring amino acid. A "substitution" refers to the replacement of one or more amino acid residue (s) by another amino acid residue (s) in the polypeptide. Derivatives can contain different combinations of alterations including more than one alteration and different types of alterations.

While the effect of an amino acid change varies depending upon factors such as phosphorylation, glycosylation, intra-chain linkages, tertiary structure, and the role of the amino acid in the active site or a possible allosteric site, it is generally preferred that the substituted amino acid is from the same group as the amino acid being replaced. To some extent the following groups contain amino acids which are interchangeable: the basic amino acids lysine, arginine, and histidine; the acidic amino acids aspartic and glutamic acids; the neutral polar amino acids serine, threonine, cysteine, glutamine, asparagine and, to a lesser extent, methionine; the nonpolar aliphatic amino acids glycine, alanine, valine, isoleucine, and leucine (however, because of size, glycine and alanine are more closely related and valine, isoleucine and leucine are more closely related) ; and the aromatic amino acids phenylalanine, tryptophan, and tyrosine. In addition, although classified in different categories, alanine, glycine, and serine seem to be interchangeable to some extent, and cysteine additionally fits into this group, or may be classified with the polar neutral amino acids.

While proline is a nonpolar neutral amino acid, its replacement represents difficulties because of its effects on conformation. Thus, substitutions by or for proline are not preferred, except when the same or similar conformational results can be obtained. The conformation conferring properties of proline residues may be obtained if one or more of these is substituted by hydroxyproline (Hyp) . Examples of modified amino acids include the following: altered neutral nonpolar amino acids such as ω-amino acids of the formula H₂N (CH₂) _nCOOH where n is 2-6, sarcosine (Sar) , t- butylalanine (t-BuAla) , t-butylglycine (t-BuGly) , N-methyl isoleucine (N-Melle) , and norleucine (Nleu) ; altered neutral aromatic amino acids such as phenylglycine; altered polar, but neutral amino acids such as citrulline (Cit) and methionine sulfoxide (MSO) ; altered neutral and nonpolar amino acids such as cyclohexyl alanine (Cha) ; altered acidic amino acids such as cysteic acid (Cya) ; and altered basic amino acids such as ornithine (Orn) .

Preferred derivatives have one or more amino acid alteration (s) which do not significantly affect the receptor activity of the related receptor protein. In regions of the GABA_BR2 not necessary for receptor activity amino acids may be deleted, added or substituted with less risk of affecting activity. In regions required for receptor activity, amino acid alterations are less preferred as there is a greater risk of affecting receptor activity. Such alterations should be conservative alterations. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent.

Conserved regions tend to be more important for protein activity than non-conserved regions. Standard procedures can be used to determine the conserved and non-conserved regions important of receptor activity using in vitro mutagenesis techniques or deletion analyses and measuring receptor activity as described by the present disclosure.

Derivatives can be produced using standard chemical techniques and recombinant nucleic acid techniques. Modifications to a specific polypeptide may be deliberate, as through site-directed mutagenesis and amino acid substitution during solid-phase synthesis, or may be accidental such as through mutations in hosts which produce the polypeptide. Polypeptides including derivatives can be obtained using standard techniques such as those described by Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory Press (1989) . For example, Chapter 15 of Sambrook describes procedures for site- directed mutagenesis of cloned DNA.

GABA_BR2 Antibodies

Antibodies binding GABA_BR2 have various uses such as being used as therapeutic agents to modulate GABA_BR activity; as diagnostic tools for determining GABA_BR2 number; as research tools for studying receptor synthesis, structure, and function; and as a tool by purifying GABA_BR2.

GABA_BR2, and GABA_BR2 fragments retaining antigenic determinants, can be used to generate antibodies recognizing GABA_BR2. Preferably, polypeptide fragments used to generate antibodies are at least six amino acid in length. Both polyclonal and monoclonal antibodies can be generated.

Antibodies can be produced using standard techniques such as those described by Harlow and Lane in Antibodies , a Labora tory- Manual , Cold Spring Harbor Laboratory, 1988. Sources of immunogens for antibody production include purified GABA_BR2, GABA_BR2 fragments, and whole cells expressing GABA_BR2. The present invention also includes hybridoma cells secreting monoclonal antibodies to GABA_BR2.

Recombinant Cells

Nucleic acid expressing a functional GABA_BR2 can be used to create transfected cells lines functionally expressing GABA_BR2. Such cell lines have a variety of uses such as being used for high-throughput screening for compounds modulating GABA_BR activity; being used to assay binding to GABA_BR2; and as factories to produce large amounts of GABA_BR2, or GABA_BR2 fragments.

A variety of cell lines can couple exogenously expressed receptors to endogenous functional responses. Cell lines such as NIH-3T3, HeLa, NG115, CHO, HEK 293 and C0S7 which are expected to lack GABA_BR2 can be tested to confirm that they lack an endogenous

GABA_BR2.

Production of stable transfectants can be accomplished by transfection of an appropriate cell line with an expression vector, such as the eukaryotic pMSG vectors. Expression vectors containing a promoter region, such as the mouse mammary tumor virus promoter (MMTV) , drive high-level transcription of cDNAs in a variety of mammalian cells. In addition, these vectors contain genes for selecting cells stably expressing cDNA of interest. The selectable marker in the pMSG vectors encodes an enzyme, xanthine-guanine phosphoribosyl transferase (XGPRT) , conferring resistance to a metabolic inhibitor that is added to the culture to kill nontransfected cells.

The most effective method for transfection of eukaryotic cell lines with plasmid DNA varies with the given cell type. The GABA_BR2 expression construct will be introduced into cultured cells by the appropriate technique, such as Ca²⁺ phosphate precipitation, DEAE-dextran transfection, lipofection or electroporation. Expression of the GABA_BR2 cDNA in cell lines can be assessed by solution hybridization and Northern blot analysis.

Assaying For Compounds Modulating GABA_BR Activity

The ability of compounds to modulate GABA_BR activity can be assayed by measuring alterations of cellular processes affected by GABA_BR activity. Generally, a GABA_BR2 agonist is present when measuring antagonist activity. However, protein fusions can be created, for example, where an agonist extracellular binding domain of GABA_BR2 is swapped with the agonist binding domain of a different receptor allowing for the measurement of antagonist activity using an agonist of the different receptor; or where the intracellular domain of GABA_BR2 is swapped with the intracellular domain of a different receptor allowing for the measuring of GABA_BR activity by measuring intracellular effects caused by the different receptor. Chimeric proteins are preferably produced using recombinant nucleic acid techniques to provide an appropriate nucleic acid encoding for the chimeric protein. Preferably, portions of GABA_BR2 are swapped with portions of the calcium receptor. The GABA_BR2 extracellular domain is made up of approximately amino acids 1-422 Of SEQ. ID. NO. 4, the GABA_BR2 transmembrane domain is made up of approximately amino acids 423-686 Of SEQ. ID. NO. 4, and the GABA_BR2 intracellular domain is made up of approximately amino acids 687-883 Of SEQ. ID. NO. 4. The human calcium receptor amino acid and encoding nucleic acid is provided in

Figure 3. The calcium receptor extracellular domain is made up of approximately amino acids 1-612, the calcium receptor transmembrane domain is made up of approximately amino acids 613-

862, and the calcium receptor intracellular domain is made up of approximately amino acids 863-1078. Calcium receptor activity can be measured using techniques well known in the art such as those described by Brown et al . , U.S. Patent No. 5,688,938, hereby incorporated by reference herein.

Binding Assays

The present invention also includes using GABA_BR2 and fragments thereof in binding assays . Binding assays can be carried out using techniques well known in the art. Binding assays preferably employ radiolabeled binding agents. An example of a binding assay is carried out by first attaching GABA_BR2, or a fragment thereof, to a solid-phase support to create an affinity matrix. The affinity matrix is then contacted with potential GABA_BR2 binding agents. A large library of compounds may be used to determine those compounds binding to the affinity matrix. Bound compounds can be eluted from the column.

Transgenic Animals

The present invention also concerns the construction and use of transgenic animals, and transformed cells, encoding GABA_BR2.

Transgenic nonhuman mammals are particularly useful as an in vivo test system for studying the effects of introducing GABA_BR2; regulating the expression of GABA_BR2 (e.g., through the introduction of additional genes, antisense nucleic acids, or ribozymes) ; and studying the effect of compounds which mimic or block the effect of GABA_BR2.

Experimental model systems for studying the physiological role of the GABA_BR2 can be created having varying degrees of receptor expression. For example, nucleic acid encoding a receptor may be inserted into cells naturally expressing the receptor such that the gene is expressed at much higher levels.

Alternatively, a recombinant gene may be used to inactivate the endogenous gene by homologous recombination and, thereby, create an GABA_BR2 deficient cell, tissue, or animal.

Inactivation of a gene can be caused, for example, by using a recombinant gene engineered to contain an insertional mutation

(e.g., the neo gene) . The recombinant gene is inserted into the genome of a recipient cell, tissue or animal, and inactivates transcription of the receptor. Such a construct may be introduced into a cell, such as an embryonic stem cell, by techniques such as transfection, transduction, and injection. Stem cells lacking an intact receptor sequence may generate transgenic animals deficient in the receptor.

Preferred test models are transgenic animals. A transgenic animal has cells containing DNA which has been artificially inserted into a cell and inserted into the genome of the animal which develops from that cell. Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats .

A variety of methods are available for producing transgenic animals. For example, DNA can be injected into the pronucleus of a fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo) following the initiation of cell division (Brinster et al . , Proc . Na t . Acad. Sci . USA 82 : 4438- 4442, 1985) . By way of another example, embryos can be infected with viruses, especially retroviruses, modified to carry GABA_BR2 nucleotide sequences.

Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention. A transgenic animal can be produced from such stem cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA) , Taconic (Germantown, NY) , and Harlan Sprague Dawley (Indianapolis, IN) . Methods for the culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct injection are well known to those of ordinary skill in the art. See, for example, Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E.J. Robertson, ed., IRL Press (1987).

Procedures for embryo manipulations are well known in the art. Procedures for manipulating rodent embryo and for microinjecting DNA into the pronucleus of the zygote are well known in the art. Microinjection procedures for fish, amphibian eggs and birds are well known in the art and are described, for example, in Houdebine and Chourrout, Experientia 41 : 897-905, 1991. Procedures for introducing DNA into tissues of animals are well known in the art and are described, for example, in U.S. Patent No. 4, 945,050.

Transfection and isolation of desired clones can be carried out using standard techniques (e.g., E.J. Robertson, supra) . For example, random gene integration can be carried out by co- transfecting nucleic acid with a gene encoding antibiotic resistance. Alternatively, for example, the gene encoding antibiotic resistance is physically linked to a nucleic acid sequence encoding GABA_BR2.

DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination. {E. g. , Capecchi, Science 244 : 1288-1292, 1989.) Methods for positive selection of the recombination event (e.g., neomycin resistance) and dual positive-negative selection (e.g., neomycin resistance and gancyclovir resistance) and the subsequent identification of the desired clones by PCR have been described in references such as Capecchi, supra and Joyner et al . , Na ture 338:153-156, 1989, which is hereby incorporated by reference herein.

The final phase of the procedure is to inject targeted ES cells into blastocysts and to transfer the blastocysts into pseudopregnant females. The resulting chimeric animals are bred and the offspring are analyzed by Southern blotting to identify individuals carrying the transgene.

An example describing the preparation of a transgenic mouse is as follows. Female mice are induced to superovulate and placed with males. The mated females are sacrificed by C0₂ asphyxiation or cervical dislocation and embryos are recovered from excised oviducts. Surrounding cumulus cells are removed. Pronuclear embryos are then washed and stored until the time of injection.

Randomly cycling adult female mice paired with vasectomized males serve as recipients for implanted embryos. Recipient females are mated at the same time as donor females and embryos are transferred surgically to recipient females.

Procedures for generating transgenic rats are similar to that of mice. {E. g. , Hammer et al . , Cell 63:1099-1112, 1990.) Procedures for producing transgenic non-rodent mammals and other animals are well known in art. {E . g. , Houdebine and Chourrout, supra ; Pursel et al . , Science 244:1281-1288, 1989; and Simms et al . , Bio/Technology 5:179-183, 1988.)

Therapeutic Modulation

Different types of diseases and disorders can be treated using compounds modulating GABA_BR activity. Additionally, such compounds can be used prophylactically . Compounds modulating GABA_BR activity can be administered to patients who would benefit from such treatment. Patients are mammals, preferably humans.

Modulating GABA_BR activity can be carried to achieve useful therapeutic effects such as preventing or treating one or more of the following: spasticity and motor control disorders using GABA_BR agonists; pain, using GABA_BR antagonists; cognitive disorders using GABA_BR antagonists; neurological disorders such as Alzheimer' s disease and Huntington' s disease; psychiatric disorders, such as depression using GABA_BR agonists; alcohol dependence and withdrawal using GABA_BR antagonists; feeding behavior; cardiovascular and respiratory disorders with antagonists exerting an excitatory effect and agonists depressing inspiratory neurons; and peripheral function disorders. Modulators of GABA_BR activity can be administered to a patient using standard techniques. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, 18^th ed., Mack Publishing Co., Easton, PA, 1990 (hereby incorporated by reference herein) . Suitable dosage forms, in part, depend upon the use or the route of entry, for example, oral, transdermal, transmucosal, or by injection (parenteral) . Such dosage forms should allow the therapeutic agent to reach a target cell whether the target cell is present in a multicellular host or in culture. For example, pharmacological compounds or compositions injected into the blood stream should be soluble. Other factors are well known in the art, and include considerations such as toxicity and dosage forms which retard the therapeutic agent from exerting its effect. Therapeutic compounds can be formulated as pharmaceutically acceptable salts and complexes thereof. Pharmaceutically acceptable salts are non-toxic salts in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of the compound without preventing it from exerting its physiological effect. Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.

The pharmaceutically acceptable salt of a compound may be present as a complex. Examples of complexes include an 8- chlorotheophylline complex (analogous to, e.g., dimenhydrinate :diphenhydramine 8-chlorotheophylline (1:1) complex; Dramamine) and various cyclodextrin inclusion complexes. Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, cyclohexylsulfa ate and quinate .

Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfa ic acid, fumaric acid, and quinic acid. Pharmaceutically acceptable salts also include basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglu ine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional groups, such as carboxylic acid or phenol are present. For example, see

Remington's Pharmaceutical Sciences, 18^th ed., Mack Publishing Co., Easton, PA, p. 1445, 1990. Such salts can be prepared using the appropriate corresponding bases.

Carriers or excipients can also be used to facilitate administration of therapeutic agents. Examples of carriers include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile solutions of water for injection (WFI), saline solution and dextrose. GABA_BR modulating compounds can be administered by different routes including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal) , or transmucosal administration. For systemic administration, oral administration is preferred. For oral administration, for example, the compounds can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.

Alternatively, injection (parenteral administration) may be used, e.g., intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, compounds are formulated in liquid solutions, preferably, in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced.

Systemic administration can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are well known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration, for example, may be through nasal sprays, rectal suppositories, or vaginal suppositories. For topical administration, compounds can be formulated into ointments, salves, gels, or creams, as is well known in the art.

The amounts of various GABA_BR modulating compounds to be administered can be determined by standard procedures taking into account factors such as the compound IC₀, EC₅₀, the biological half-life of the compound, the age, size and weight of the patient, and the disease or disorder associated with the patient. The importance of these and other factors to be considered are well known to those of ordinary skill in the art. Generally, the amount is expected to preferably be between about 0.01 and 50 mg/kg of the animal to be treated.

EXAMPLES The example provided below illustrates different aspects and embodiments of the present invention. The example is not intended to limit the claimed invention.

Functional expression of GABA„R2

Xenopus oocytes were co-injected with in vi tro transcribed RNA (7 ng) encoding GABA_BRla, GABA_BR2 and chimeric Gqo5. Chimeric Gqo5 is described in Na ture 363 : 214-276, 1993. Coexpression of the different proteins was employed because GABA_BR functions as a heterodimer of the subunits GABA_BR1 or GABA_BR2 (Jones et al . Na ture 396: 614-619 , 1998). Following a 72 hour incubation, the oocytes were voltage clamped using standard electrophysiological techniques (Hille, B., Ionic Channels of Excitable membranes, pp. 30-33, Sinauer Associates, Inc., Sunderland, MA, 1992). Activation of the receptor heterodimers was detected by increases in the calcium-activated chloride current. Application of the GABA_B receptor agonist baclofen caused dose-dependent, reversible, oscillatory increases in the calcium- activated chloride current as shown in Figure 4, with an EC₅₀ of approximately 1 μM. These responses were completely blocked by the competitive GABA_B receptor antagonist SCH 50911 (100 μM) . Oocytes expressing GABA_B receptor heterodimers with the inwardly rectifying potassium channels (GIRKS; Kir3.1/3.2/3.4 ) were used as the positive control (Jones et al . , Na ture 396: 614- 619 , 1998.) Thus, the use of the chimeric G-Protein Gqo5 promotes signal transduction through mobilization of intracellular calcium. Other embodiments are within the following claims. Thus, while several embodiments have been shown and described, various modifications may be made, without departing from the spirit and scope of the present invention.

Claims

Claims

1. A purified nucleic acid comprising at least 18 contiguous nucleotides of a nucleic acid sequence provided in SEQ ID NO: 1.

2. The purified nucleic acid of claim 1, comprising at least 27 contiguous nucleotides of the nucleic acid sequence provided in SEQ ID NO: 1.

3. The purified nucleic acid of claim 2, comprising at least 45 contiguous nucleotides of the nucleic acid sequence provided in SEQ ID NO: 1.

4. The purified nucleic acid of claim 3, comprising the nucleic acid sequence provided in SEQ ID NO: 1.

5. A purified nucleic acid comprising a nucleic acid sequence encoding at least 6 contiguous amino acids of an amino acid sequence provided in SEQ. ID. NO: 4.

6. The purified nucleic acid of claim 5, wherein said nucleic acid encodes at least 12 contiguous amino acids of the amino acid sequence provided in SEQ. ID. NO: 4.

7. The purified nucleic acid of claim 6, wherein said nucleic acid encodes at least 18 contiguous amino acids of the amino acid sequence provided in SEQ. ID. NO: 4.

8. The purified nucleic acid of claim 7, wherein said nucleic acid encodes at least 54 contiguous amino acids of the amino acid sequence provided in SEQ. ID. NO: 4.

9. The purified nucleic acid of claim 8, wherein said nucleic acid encodes the amino acid sequence provided in SEQ. ID. NO: 4.

10. The purified nucleic acid of any of claims 1-9, wherein said nucleic acid is substantially purified.

11. The purified nucleic acid of any of claims 1-9, wherein said nucleic acid is recombinant nucleic acid which is part of an expression vector.

12. The purified nucleic acid of any of claims 1-9, wherein said nucleic acid is transcriptionally coupled to an exogenous promoter.

13. A recombinant cell comprising the expression vector of claim 11.

14. A recombinant cell made by a process comprising the step of introducing the nucleic acid of any one of claims 1-12 into a cell.

15. A purified nucleic acid comprising a nucleotide sequence of 20 contiguous nucleotides of which at least 18 nucleotides are complementary to the nucleic acid sequence provided in SEQ ID NO: 1 or the perfect complement of SEQ ID NO: 1.

16. The nucleic acid of claim 15, wherein said purified nucleic acid comprises a nucleotide sequence of 20 contiguous nucleotides which has at least 19 bases complementary to the nucleic acid sequence provided in SEQ ID NO: 1 or the perfect complement of SEQ ID NO: 1.

17. The nucleic acid of claim 16, wherein said purified nucleic acid comprises a nucleotide sequence of 20 contiguous nucleotides which is complementary to the nucleic acid sequence provided in SEQ ID NO: 1 or the perfect complement of SEQ ID NO: 1.

18. A purified polypeptide comprising at least 6 contiguous amino acids of an amino acid sequence provided in SEQ. ID. NO: 4.

19. The purified polypeptide of claim 18, comprising at least 12 contiguous amino acids of the amino acid sequence provided in SEQ. ID. NO: 4.

20. The purified polypeptide of claim 19, comprising at least 18 contiguous amino acids of the amino acid sequence provided in SEQ. ID. NO: 4.

21. The purified polypeptide of claim 20, comprising at least 54 contiguous amino acids of the amino acid sequence provided in SEQ. ID. NO: 4.

22. The purified polypeptide of claim 21, consisting of the amino acid sequence provided in SEQ. ID. NO: 4.

23. The polypeptide of any one of claims 18-22, wherein said polypeptide is substantially purified.

24. A purified GABA_BR2-binding agent comprising a molecule which binds to a polypeptide consisting of the amino acid sequence of SEQ. ID. NO: 4.

25. The binding agent of claim 24, wherein said binding agent is an antibody.

26. A method of making a GABA_BR2 or fragment thereof comprising the step of incubating the recombinant cells of claim 13 under conditions wherein the nucleic acid encoding for the GABA_BR2 is expressed.

27. The method of claim 26, further comprising the step of purifying said GABA_BR2 or fragment thereof.

28. A method of selecting for a compound modulating GABA_BR activity comprising the steps of a) contacting a recombinant cell functionally expressing GABA_BR2 with a first test compound; and b) measuring the ability of said test compound to affect GABA_BR activity to select for said compound modulating GABA_BR activity .

29. The method of claim 28, wherein the ability of a plurality of different test compounds to affect GABA_BR activity are tested to select for said compound modulating GABA_BR activity.

30. A coexpression system comprising a) a cell; b) at least one of GABA_BRla and GABA_BRlb, which is present in said cell; c) GABA_BR2, which is present in said cell; and d) Gqo5, which is present in said cell.

31. A method of screening for one or more compounds active at GABA_BRla, GABA_BRlb, or GABA_BR2 comprising the steps of contacting the coexpression system of claim 30 with at least one of said compounds and measuring the ability of said compounds to effect the mobilization of intracellular calcium.

32. The method of claim 31, wherein 10 or more compounds are individually tested for their ability to effect the mobilization of intracellular calcium over the course of 8 hours.

33. A transgenic nonhuman mammal comprising a nonhuman mammal and a recombinant nucleic acid encoding a polypeptide comprising 6 contiguous amino acids of an amino acid sequence provided in SEQ. ID. NO: 4.