HUMAN ALPHA4 RECEPTOR SUBUNIT OF THE GABA-A RECEPTOR

Abstract: The present invention relates to the cloning of novel cDNA sequences encoding the alpha 4 and delta receptor subunits of the human GABAA receptor; to stably co-transfected eukaryotic cell lines capable of expressing a human GABAA receptor, which receptor comprises at least one of the novel alpha 4 and delta receptor subunits; and to the use of such cell lines in screening for and designing medicaments which act upon the human GAGAA receptor.

Description: HUMAN ALPHA 4 RECEPTOR SUBUNIT OF THE GABA-A RECEPTOR This invention concerns the cloning of a novel cDNA sequence encoding a particular subunit of the human GABA^ receptor. In addition, the invention relates to a stable cell line capable of expressing said cDNA and to the use of the cell line in a screening technique for the design and development of subtype-specific medicaments. Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. It mediates fast synaptic inhibition by opening the chloride channel intrinsic to the GABA^ receptor. This receptor comprises a multimeric protein of molecular size 230-270 kDa with specific binding sites for a variety of drugs including benzodiazepines, barbiturates and β-carbolines, in addition to sites for the agonist ligand GABA (for reviews see Stephenson, Biochem. J., 1988, 249, 21; Olsen and Tobin, Faseb J., 1990, 4, 1469; and Sieghart, Trends in Pharmacol. Sci., 1989, 10, 407). Molecular biological studies demonstrate that the receptor is composed of several distinct types of subunit, which are divided into four classes (α, β, γ and δ) based on their sequence similarities. To date, six types of α (Schofield et al., Nature (London), 1987, 328, 221; Levitan et al., Nature (London), 1988, 335, 76; Ymer et al., EMBO J., 1989, 8, 1665; Pritchett & Seeberg, J. Neurochem., 1990, 54, 802; Luddens et al., Nature (London), 1990, 346, 648; and Khrestchatisky et al, Neuron, 1989, 3, 745), three types of β (Ymer et al., EMBO J., 1989, 8, 1665), three types of γ (Ymer et al., EMBO J., 1990, 9, 3261; Shivers et al., Neuron, 1989, 3, 327; and Knoflach et al, FEBS Lett., 1991, 293, 191) and one δ subunit (Shivers et al., Neuron, 1989, 3, 327) have been identified. The differential distribution of many of the subunits has been characterised by in situ hybridisation (Sequier et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 7815; Malherbe et al., J. Neurosci., 1990, 10, 2330; Shivers et al, Neuron, 1989, 3, 327; and Wisden et al, J. Neurosci., 1992, 12, 1040) and this has permitted it to be speculated which subunits, by their co-localisation, could theoretically exist in the same receptor complex. Various combinations of subunits have been co-transfected into cells to identify synthetic combinations of subunits whose pharmacology parallels that of bona fide GABA^ receptors in vivo (Pritchett et al, Science, 1989, 245, 1389; Malherbe et al, J. Neurosci., 1990, 10, 2330; Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802; and Luddens et α/., Nature (London), 1990, 346, G48). This approach has revealed that, in addition to an α and β subunit, either γi or γ (Pritchett et al, Nature (London), 1989, 338, 582; Ymer et al, EMBO J., 1990, 9, 3261; and Malherbe et al, J. Neurosci., 1990, 10, 2330) or 73 (Herb et al, Proc. Natl Acad. Sci. USA, 1992, 89, 1433; Knoflach et al, FEBS Lett., 1991, 293, 191; and Wilson-Shaw et αZ., FEBS Lett., 1991, 284, 211) is also generally required to confer benzodiazepine sensitivity, and that the benzodiazepine pharmacology of the expressed receptor is largely dependent on the identity of the α and γ subunits present. Receptors containing a δ subunit (i.e. αβδ) do not appear to bind benzodiazepines (Shivers et al, Neuron, 1989, 3, 327). Combinations of subunits have been identified which exhibit the pharmacological profile of a BZ \ type receptor (oqβ 1 2) an d a BZ2 type receptor (o-2βιγ2 or a 33lY2> Pritchett et al, Nature (London), 1989, 338, 582), as well as two GABA^ receptors with a novel pharmacology, o^2Y2 (Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802) and o^2Y2 (Luddens et al, Nature (London), 1990, 346, 648). Although the pharmacology of these expressed receptors appears similar to that of those identified in brain tissue by radioligand binding, it has nonetheless not been shown that these receptor subunit combinations exist in viυo. A combination of subunits comprising either the human 0-4 GABA^ receptor subunit and/or the δ GABA^ receptor subunit has not hitherto been possible due to the non-availability of the human a.4 cDNA or human δ cDNA. This has consequently limited the use of cell lines in screening for subtype-specific medicaments, it being impossible to study the pharmacological profile of subunit combinations comprising the 014 subunit and/or the δ subunit. We have now ascertained the cDNA sequence of the c.4 subunit and the δ subunit of the human GABAA receptor. These nucleotide sequences, together with their deduced amino acid sequences corresponding thereto, are depicted in Figures 2 and 3 of the accompanying drawings. The present invention accordingly provides, in a first aspect, a DNA molecule encoding the 0:4 subunit of the human GABA^ receptor comprising all or a portion of the sequence depicted in Figure 2, or a modified human sequence. The present invention also provides, in another aspect, a DNA molecule encoding the δ subunit of the human GABA^ receptor comprising all or a portion of the sequence depicted in Figure 3, or a modified human sequence. The sequencing of the novel cDNA molecules in accordance with the invention can conveniently be carried out by the standard procedure described in accompanying Example 1; or may be accomplished by alternative molecular cloning techniques which are well known in the art, such as those described by Maniatis et al. in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York, 2nd edition, 1989. In another aspect, the invention provides a recombinant expression vector comprising the nucleotide sequence of the human GABA^ receptor α.4 subunit together with additional sequences capable of directing the synthesis of the said human G BAA receptor 0:4 subunit in cultures of stably co-transfected eukaryotic cells. The present invention also provides a recombinant expression vector comprising the nucleotide sequence of the human GABA^ receptor δ subunit together with additional sequences capable of directing the synthesis of the said human GABA^ receptor δ subunit in cultures of stably co-transfected eukaryotic cells. The term "expression vectors" as used herein refers to DNA sequences that are required for the transcription of cloned copies of recombinant DNA sequences or genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, blue-green algae, yeast cells, insect cells, plant cells and animal cells. Specifically designed vectors allow the shuttling of DNA between bacteria-yeast, bacteria-plant or bacteria- animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selective markers, a limited number of useful restriction enzyme sites, a high copy number, and strong promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and to initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. The term "cloning vector" as used herein refers to a DNA molecule, usually a small plasmid or bacteriophage DNA capable of self-replication in a host organism, and used to introduce a fragment of foreign DNA into a host cell. The foreign DNA combined with the vector DNA constitutes a recombinant DNA molecule which is derived from recombinant technology. Cloning vectors may include plasmids, bacteriophages, viruses and cosmids. The recombinant expression vector in accordance with the invention may be prepared by inserting the nucleotide sequence of the GABA^ 0.4 subunit or the GABA^ δ subunit into a suitable precursor expression vector (hereinafter referred to as the "precursor vector") using conventional recombinant DNA methodology known from the art. The precursor vector may be obtained commercially, or constructed by standard techniques -from known expression vectors. The precursor vector suitably contains a selection marker, typically an antibiotic resistance gene, such as the neomycin or ampicil-in resistance gene. The precursor vector preferably contains a neomycin resistance gene, adjacent the SV40 early splicing and polyadenylation region; an ampicillin resistance gene; and an origin of replication, e.g. pBR322 ori. The vector also preferably contains an inducible promoter, such as MMTV-LTR (inducible with dexamethasone) or metallothionin (inducible with zinc), so that transcription can be controlled in the cell line of this invention. This reduces or avoids any problem of toxicity in the cells because of the chloride channel intrinsic to the GABA^ receptor. One suitable precursor vector is pMAMneo, available from Clontech Laboratories Inc. (Lee et al, Nature, 1981, 294, 228; and Sardet et al, Cell, 1989, 56, 271). Alternatively the precursor vector pMSGneo can be constructed from the vectors pMSG and pSV2neo. The recombinant expression vector of the present invention is then produced by cloning the GABA^ receptor 0-4 subunit cDNA or the GABA^ receptor δ subunit cDNA into the above precursor vector. The receptor subunit cDNA is subcloned from the vector in which it is harboured, and ligated into a restriction enzyme site, e.g. the Hind III site, in the polylinker of the precursor vector, for example pMAMneo or pMSGneo, by standard cloning methodology known from the art, and in particular by techniques analogous to those described herein. Before this subcloning, it is often advantageous, in order to improve expression, to modify the end of the 0:4 or δ subunit cDNA with additional 5' untranslated sequences, for example by modifying the 5' end of the 0-4 or δ subunit DNA by addition of 5' untranslated region sequences from the o subunit DNA. One suitable expression vector of the present invention is illustrated in Fig. 1 of the accompanying drawings, in which R represents the nucleotide sequence of the 04 or δ subunit of the GABAA receptor, and the remainder of the expression vector depicted therein is derived from the precursor vector pMSGneo. According to a further aspect of the present invention, there is provided a stably co-transfected eukaryotic cell line capable of expressing a GABAA receptor, which receptor comprises the alpha-4 receptor subunit, at least one beta receptor subunit and the delta receptor subunit. In another aspect of the present invention, there is provided a stably co-transfected eukaryotic cell line capable of expressing a GABA^ receptor, which receptor comprises the alpha-4 receptor subunit, at least one beta receptor subunit and at least one gamma receptor subunit. In a further aspect of the present invention, there is provided a stably co-transfected eukaryotic cell line capable of expressing a GABA^. receptor, which receptor comprises at least one alpha receptor subunit, at least one beta receptor subunit and the delta receptor subunit. This is achieved by co-transfecting cells with three expression vectors, each harbouring cDNAs encoding for an 0:4, β or δ GABA^ receptor subunit, or for an 1x4, β or γ GABA^ receptor subunit, or for an α, β or δ GABAA receptor subunit. In a further aspect, therefore, the present invention provides a process for the preparation of a eukaryotic cell line capable of expressing a GABA^ receptor, which comprises stably co-transfecting a eukaryotic host cell with at least three expression vectors, one such vector harbouring the cDNA sequence encoding the 014 GABAA receptor subunit another such vector harbouring the cDNA sequence encoding a beta GABA^ receptor subunit, and a third such vector harbouring the cDNA sequence encoding the delta GABA^ receptor subunit. The stable cell-line which is established expresses an o-4βδ GABAA receptor. The present invention also provides a process for the preparation of a eukaryotic cell line capable of expressing a GABA^ receptor, which comprises stably co-transfecting a eukaryotic host cell with at least three expression vectors, one such vector harbouring the cDNA sequence encoding the 04 GABA^ receptor subunit another such vector harbouring the cDNA sequence encoding a beta GABA^ receptor subunit, and a third such vector harbouring the cDNA sequence encoding a gamma GABA^ receptor subunit. The stable cell-line which is established expresses an oi4βγ GABAA receptor. Similarly, the present invention provides a process for the preparation of a eukaryotic cell line capable of expressing a GABA^ receptor, which comprises co-transfecting a eukaryotic host cell with at least three expression vectors, one such vector harbouring the cDNA sequence encoding an alpha GABA^ receptor subunit, another such vector harbouring the cDNA sequence encoding a beta GABA^ receptor subunit, and a third such vector harbouring the cDNA sequence encoding the δ GABAA receptor subunit. The stable cell line which is established expresses an αβδ GABA^ receptor. Each receptor thereby expressed, comprising a unique combination of 0:4, β and δ subunits, or 04, β and γ subunits, or α, β and δ subunits, will be referred to hereinafter as a GABA^ receptor "subunit combination". Pharmacological and electrophysiological data confirm that the recombinant α4βγ receptor expressed by the cells of the present invention has the properties expected of a native GABA^ receptor. Expression of the GABA^ receptor may be accomplished by a variety of different promoter-expression systems in a variety of different host cells. The eukaryotic host cells suitably include yeast, insect and mammalian cells. Preferably the eukaryotic cells which can provide the host for the expression of the receptor are mammahan cells. Suitable host cells include rodent fibroblast lines, for example mouse Ltk", Chinese hamster ovary (CHO) and baby hamster kidney (BHK); HeLa; and HEK293 cells. It is necessary to incorporate the α.4 subunit, at least one β and the δ subunit into the cell line in order to produce the required receptor, or alternatively the 0:4 subunit and at least one β and one γ subunit or alternatively at least one α, one β and the δ subunit. Within this limitation, the choice of receptor subunit combination is made according to the type of activity or selectivity which is being screened for. In order to employ this invention most effectively for screening purposes, it is preferable to build up a library of cell lines, each with a different combination of subunits. Typically a library of 5 or 6 cell line types is convenient for this purpose. Preferred subunit combinations include: 0^372, o-4β3δ and αgβ3δ. Another preferred subunit combination is c-^2Y2* As stated above, for each cell line of the present invention, three such vectors will be necessary, one containing the 04 subunit, one containing a β subunit, and the third containing the δ subunit, or alternatively, one containing the 0.4 subunit, one containing a β subunit, and the third containing a γ subunit, or alternatively, one containing an α subunit, one containing a β subunit and one containing the δ subunit. Cells are then co-transfected with the desired combination of three expression vectors. There are several commonly used techniques for transfection of eukaryotic cells in vitro. Calcium phosphate precipitation of DNA is most commonly used (Bachetti et al, Proc. Natl. Acad. Sci. USA, 1977, 74, 1590-1594; Maitland e- al., Cell, 1977, 14, 133-141), and represents a favoured technique in the context of the present invention. A small percentage of the host cells takes up the recombinant DNA. In a small percentage of those, the DNA will integrate into the host cell chromosome. Because the neomycin resistance gene will have been incorporated into these host cells, they can be selected by isolating the individual clones which will grow in the presence of neomycin. Each such clone is then tested to identify those which will produce the receptor. This is achieved by inducing the production, for example with dexamethasone, and then detecting the presence of receptor by means of radioligand binding. In a further aspect, the present invention provides protein preparations of GABA^ receptor subunit combinations, especially human GABA^ receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells. The invention also provides preparations of membranes containing subunit combinations of the GABA^ receptor, especially human GABA^ receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells. The cell line, and the membrane preparations therefrom, according to the present invention have utility in screening and design of drugs which act upon the GABAA receptor, for example benzodiazepines, barbiturates, β-carbolines and neurosteroids. The present invention accordingly provides the use of the cell line described above, and membrane preparations derived therefrom, in screening for and designing medicaments which act upon the GABA^ receptor. Of particular interest in this context are molecules capable of interacting selectively with GABAA receptors made up of varying subunit combinations. As will be readily apparent, the cell line in accordance with the present invention, and the membrane preparations derived therefrom, provide ideal systems for the study of structure, pharmacology and function of the various GABAA receptor subtypes. The following non-limiting Examples illustrate the present invention. EXAMPLE 1 ISOLATION AND SEQUENCING OF cDNAS ENCODING THE HUMAN GABAA RECEPTOR α SUBUNIT a) cDNA libraries cDNAs were cloned from human foetal brain and adult hippocampus cDNA libraries. All cDNA libraries were constructed in the lambdaZAP vector, and were purchased from Stratagene (San Diego, California). For screening, the cDNA libraries were plated according to the manufacturer's instructions, at 40,000 pfu per 137 mm plate. Filter lifts were taken using Hybond N filters (Amersham) according to the manufacturer's instructions. b) Isolation of cDNA encoding human 04 subunit A human 04 probe was first generated by polymerase chain reaction (PCR) using oligonucleotide primers (synthesised on an Applied Biosystems 380B synthesizer) derived from the bovine 0:4 sequence (Ymer et al, FEBS Lett., 1989, 258, 119): 5'TTTCAGGAATTCCAGTGCTGAGAGAAAAGCATCCTGAAAC3' (bp 1121-1160, containing an EcoRI restriction enzyme site) SEQ. ID. NO.:l, and 5'ATCCAGAAGCTTGTGGAGCAGAGGGAGTAGTAGTGGC3' (antisense, bp 1540-1577, incorporating a Hindlll restriction enzyme site) SEQ. ID. NO. :2. PCR was performed as described, for example, by Whiting et al in Proc. Natl Acad. Sci., USA, 1990, 87, 9966, using a human foetal brain cDNA library as a template. The PCR product was digested with EcoRI and Hindlll and subcloned into similarly digested pBluescript SK- and its identity confirmed by DNA sequencing using standard techniques and the Sequenase II enzyme (United States Biochemicals). A human foetal brain cDNA library was screened using 3 p labelled human 0.4 probe DNA as described above. A single cDNA clone, approximately 2500bp, was obtained. DNA sequencing indicated that this cDNA clone contained 3' untranslated sequences and 3' coding region up to bp 1162 of the bovine cDNA sequence. The missing 5' sequence was obtained by anchored PCR using human brain 5'-RACE-Ready cDNA (CLONTECH, Palo Alto, CA), according to the manufacturer's instructions. The antisense oligonucleotides used for nested PCR were δ'ATTGGCATTTGTATTCTGCAGAGGS' SEQ. ID. NO.:3, and 5'GGAAGATTTGCTTGAATGGTTTGG3 * SEQ. ID. NO.:4. A 1200bp PCR product was obtained. DNA sequencing confirmed that this cDNA contained the missing 5' sequence of the 0:4 cDNA, extending to 130bp 5' of the initiating ATG codon. A full length 04 cDNA was generated by PCR using oligonucleotide primers generated from sequences of the 5' and 3' untranslated region: 5' sense primer δ'CCTGGATCCGTGAACAGGCTTGAAGTATGS' (incorporating a BamHI restriction enzyme site) SEQ. ID. NO.:5; 3' antisense primer 5'ACGAATTCACATTAGACTTTCTGATTTCTC3 * (incorporating an EcoRI restriction enzyme site) SEQ. ID. NO.:6. PCR was performed using human brain thalamus cDNA. A 1500bp product was generated which was subcloned into the cloning/eukaryotic expression vector pcDNA/Amp (Invitrogen). The cDNA was sequenced completely on both strands using an Applied Biosystems 373A DNA sequencer and dye terminator chemistry according to the manufacturer's instructions. The complete nucleotide sequence of the cDNA encoding the human 014 subunit, together with the deduced amino acid sequence corresponding thereto is shown in Fig. 2 of the accompanying drawings SEQ. ID. NOS.:7 and 8. EXAMPLE 2 ISOLATION AND SEQUENCING OF cDNAS ENCODING THE HUMAN GABA A RECEPTOR δ SUBUNIT a) cDNA libraries As described in Example 1(a). b) Isolation of cDNA encoding human δ subunit A rat δ subunit probe was first generated by PCR using oligonucleotide primers derived from the rat δ subunit sequence (Shivers et al, Neuron, 1989, 3, 327): 5*AGCCCGAATTCCATGGACGTTCTGGGCTGGCTG3' (bp 18-51, incorporating an EcoRI restriction enzyme site) SEQ. ID. NO.:9 and 5 , GGTTTCCAAGCTTACTTTGGAGAGGTAGC3 , (bp 1357-1390, incorporating a Hindlll restriction enzyme site) SEQ. ID. NO.: 10. PCR was performed as described, for example, by Whiting et al, Proc. Natl. Acad. Sci., USA, 1990, 87, 9966, using rat brain cDNA as template. A 1400bp product was obtained, subcloned into pBluescript SK- and its identity confirmed by DNA sequencing. A human hippocampus cDNA library was screened using 32p labelled rat δ subunit probe DNA as described above. A single clone was obtained containing an 1800bp insert. DNA sequencing indicated that this cDNA contained the complete coding region of the human δ subunit. The cDNA was sequenced completely on both strands using an Applied Biosystems 373A DNA sequencer and dye terminator chemistry according to the manufacturer's instructions. The complete nucleotide sequence of the cDNA encoding the human δ subunit, together with the deduced amino acid sequence corresponding thereto is shown in Fig. 3 of the accompanying drawings SEQ. ID. NOS.: l l and 12. EXAMPLE 3 EXPRESSION OF HUMAN α cDNA IN XENOPUS OOCYTES ~~ The human 0-4 cDNA (Example 1, Fig. 2) was subcloned into the eukaryotic expresion vector, pCDNA I Amp (Invitrogen, San Diego CA). Expression of this cDNA was investigated using the Xenopus oocyte system. Methods for preparation of Xenopus oocytes, nuclear injection of cDNAs, and eletrophysiological recordings from oocytes expressing recombinant GABA^ receptors, are well documented (see, for instance, Hadingham et al, Mol. Pharmacol, 1993, 44, 1211-1218). When co-expressed with β 2 and γ2 cDNAs (Hadingham et al, Mol Pharmacol, 1993, 44, 1211-1218) minimal expressed of GABA^. gated chloride currents were observed (10-50nA whole cell currents as measured under voltage clamped conditions). To increase the efficiency of expression the 0-4 cDNA was re-engineered so as to replace the 5' untranslated sequence and signal peptide with the corresponding cq sequence. PCR was performed using the cq cDNA (Schofield et al, Nature (London), 1987, 328, 221) as template. Primers were (i) 5 AATGAGTTTAAACCATAGCTTCTTCCAGT3' (bp 12-35 of αi incorporating a BamHI site) SEQ. ID. NO. : 11, and (ii) 5'CATGATGGATCCGCCCGCTCAGAC3' (bp 269-305 incorporating a Pmel site) SEQ. ID. NO.: 12. The BamHI-Pmel cut PCR fragment was subcloned into similarly cut cq. pCDNA I Amp. When this 0,4 construct was co-expressed in Xenopus oocytes with β2 and 72 cDNAs robust GABAA gated currents of up to lOOOnA whole cell current were obtained. SEQTJENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Merck Sharp & Dohme Limited (B) STREET: Terlings Park (C) CITY: Harlow (D) STATE: Essex (E) COUNTRY: England (F) POSTAL CODE (ZIP): CM20 2QR (ii) TITLE OF INVENTION: Novel Cloned GABA-A Receptor Subunit cDNA Sequences and Stably Co-transfected Cell Lines (iii) NUMBER OF SEQUENCES: 14 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTTCAGGAAT TCCAGTGCTG AGAGAAAAGC ATCCTGAAAC (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 237 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATCCAGAAGC TTGTGGAGCA GAGGGAGTAG TAGTGGC (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATTGGCATTT GTATTCTGCA GAGG (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GGAAGATTTG CTTGAATGGT TTGG (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CCTGGATCCG TGAACAGGCT TGAAGTATG (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACGAATTCAC ATTAGACTTT CTGATTTCTC (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1707 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 39..1703 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 10 20 30 41 50 59 > GGATCCGTGA ACAGCTTGAA GTATGGCATG TTGCAAAG ATG GTT TCT GCC AAG AAG GTA MET Val Ser Ala Lys Lys Val 68 77 86 95 104 113 CCC GCG ATC ACT CTG TCC GCC GGG GTC AGT TTC GCC CTC CTG CGC TTC CTG TGC Pro Ala lie Thr Leu Ser Ala Gly Val Ser Phe Ala Leu Leu Arg Phe Leu Cys 122 131 140 149 158 167 CTG GCG GTT TGT TTA AAC GAA TCC CCA GGA CAG AAC CAA AAG GAG GAG AAA TTG Leu Ala Val Cys Leu Asn Glu Ser Pro Gly Gin Asn Gin Lys Glu Glu Lys Leu 176 185 194 203 212 221 TGC ACA GAA AAT TTC ACC CGC ATC CTG GAC AGT TTG CTC GAT GGT TAT GAC AAC Cys Thr Glu Asn Phe Thr Arg lie Leu Asp Ser Leu Leu Asp Gly Tyr Asp Asn 230 239 248 257 266 275 AGG CTG CGT CCT GGA TTT GGG GGT CCT GTT ACA GAA GTG AAA ACT GAC ATA TAT Arg Leu Arg Pro Gly Phe Gly Gly Pro Val Thr Glu Val Lys Thr Asp lie Tyr 284 293 302 311 320 329 GTC ACC AGC TTT GGA CCT GTT TCT GAT GTT GAA GTG GAA TAC ACA ATG GAT GTG Val Thr Ser Phe Gly Pro Val Ser Asp Val Glu Val Glu Tyr Thr MET Asp Val 338 347 356 365 374 383 TTC TTC AGG CAG ACA TGG ATT GAC AAA AGA TTA AAA TAT GAC GGC CCC ATT GAA Phe Phe Arg Gin Thr Trp lie Asp Lys Arg Leu Lys Tyr Asp Gly Pro lie Glu 392 401 410 419 428 437 ATT TTG AGA TTG AAC AAT ATG ATG GTA ACG AAA GTG TGG ACC CCT GAT ACT TTC lie Leu Arg Leu Asn Asn MET MET Val Thr Lys Val Trp Thr Pro Asp Thr Phe 446 455 464 473 482 491 TTC AGG AAT GGA AAG AAA TCT GTC TCA CAT AAT ATG ACA GCT CCA AAT AAG CTT Phe Arg Asn Gly Lys Lys Ser Val Ser His Asn MET Thr Ala Pro Asn Lys Leu 500 509 518 527 536 545 TTT AGA ATT ATG AGA AAT GGT ACT ATT TTA TAC ACA ATG AGA CTC ACC ATA AGT Phe Arg lie MET Arg Asn Gly Thr lie Leu Tyr Thr MET Arg Leu Thr He Ser 554 563 572 561 590 599 GCG GAG TGT CCC ATG AGA TTG GTG GAT TTT CCC ATG GAT GGT CAT GCA TGC CCT Ala Glu Cys Pro MET Arg Leu Val Asp Phe Pro MET Asp Gly His Ala Cys Pro 608 617 626 635 644 653 GTG AAA TTC GGG AGT TAT GCC TAT CCA AAG AGT GAG ATG ATC TAT ACC TGG ACA Val Lys Phe Gly Ser Tyr Ala Tyr Pro Lys Ser Glu MET He Tyr Thr Trp Thr 662 671 680 689 698 707 AAA GGT CCT GAG AAA TCA GTT GAA GTT CCG AAG GAG TCT TCC AGC TTA GTT CAA Lys Gly Pro Glu Lys Ser Val Glu Val Pro Lys Glu Ser Ser Ser Leu Val Gin 716 725 734 743 752 761 TAT GAT TTG ATT GGG CAA ACC GTA TCA AGT GAA ACC ATC AAA TCA ATT ACG GGT Tyr Asp Leu He Gly Gin Thr Val Ser Ser Glu Thr He Lys Ser He Thr Gly 770 779 788 797 806 815 GAA TAT ATT GTT ATG ACG GTT TAC TTC CAC CTC AGA CGG AAG ATG GGT TAT TTT Glu Tyr He Val MET Thr Val Tyr Phe His Leu Arg Arg Lys MET Gly Tyr Phe 824 833 842 851 860 869 ATG ATT CAG ACC TAT ATT CCG TGC ATT ATG ACA GTG ATT CTT TCT CAA GTT TCA MET He Gin Thr Tyr He Pro Cys He MET Thr Val He Leu Ser Gin Val Ser 878 887 896 905 914 923 TTT TGG ATA AAT AAA GAA TCA GTT CCC GCT AGG ACC GTA TTT GGA ATA ACA ACT Phe Trp He Asn Lys Glu Ser Val Pro Ala Arg Thr Val Phe Gly He Thr Thr 932 941 950 959 968 977 GTC CTC ACC ATG ACC ACA CTA AGC ATC AGT GCA CGA CAT TCT TTG CCC AAA GTG Val Leu Thr MET Thr Thr Leu Ser He Ser Ala Arg His Ser Leu Pro Lys Val 986 995 1004 1013 1022 1031 TCC TAT GCT ACC GCC ATG GAC TGG TTC ATA GCT GTC TGC TTT GCT TTT GTA TTT Ser Tyr Ala Thr Ala MET Asp Trp Phe He Ala Val Cys Phe Ala Phe Val Phe 1040 1049 1058 1067 1076 1085 TCG GCC CTT ATC GAG TTT GCT GCT GTC AAC TAT TTC ACC AAT ATT CAA ATG GAA Ser Ala Leu He Glu Phe Ala Ala Val Asn Tyr Phe Thr Asn He Gin MET Glu 1094 1103 1112 1121 1130 1139 AAA GCC AAA AGG AAG ACA TCA AAG CCC CCT CAG GAA GTT CCC GCT GCT CCA GTG Lys Ala Lys Arg Lys Thr Ser Lys Pro Pro Gin Glu Val Pro Ala Ala Pro Val 1148 1157 1166 1175 1184 1193 CAG AGA GAG AAG CAT CCT GAA GCC CCT CTG CAG AAT ACA AAT GCC AAT TTG AAC Gin Arg Glu Lys His Pro Glu Ala Pro Leu Gin Asn Thr Asn Ala Asn Leu Asn 1202 1211 1220 1229 1238 1247 ATG AGA AAA AGA ACA AAT GCT TTG GTT CAC TCT GAA TCT GAT GTT GGC AAC AGA MET Arg Lys Arg Thr Asn Ala Leu Val His Ser Glu Ser Asp Val Gly Asn Arg 1256 1265 1274 1283 1292 1301 ACT GAG GTG GGA AAC CAT TCA AGC AAA TCT TCC ACA GTT GTT CAA GAA TCT TCT Thr Glu Val Gly Asn His Ser Ser Lys Ser Ser Thr Val Val Gin Glu Ser Ser 1310 1319 1328 1337 1346 1355 AAA GGC ACA CCT CGG TCT TAC TTA GCT TCC AGT CCA AAC CCA TTC AGC CGT GCA Lys Gly Thr Pro Arg Ser Tyr Leu Ala Ser Ser Pro Asn Pro Phe Ser Arg Ala 1364 1373 1382 1391 1400 1409 AAT GCA GCT GAA ACC ATA TCT GCA GCA AGA GCA CTT CCA TCT GCT TCT CCT ACT Asn Ala Ala Glu Thr He Ser Ala Ala Arg Ala Leu Pro Ser Ala Ser Pro Thr 1418 1427 1436 1445 1454 1463 TCT ATC CGA ACT GGA TAT ATG CCT CGA AAG GCT TCA GTT GGA TCT GCT TCT ACT Ser He Arg Thr Gly Tyr MET Pro Arg Lys Ala Ser Val Gly Ser Ala Ser Thr 1472 1481 1490 1499 1508 1517 CGT CAC GTG TTT GGA TCA AGA CTG CAG AGG ATA AAG ACC ACA GTT AAT ACC ATA Arg His Val Phe Gly Ser Arg Leu Gin Arg He Lys Thr Thr Val Asn Thr He 1526 1535 1544 1553 1562 1571 GGG GCT ACT GGG AAG TTG TCA GCT ACT CCT CCT CCA TCG GCT CCA CCA CCT TCT Gly Ala Thr Gly Lys Leu Ser Ala Thr Pro Pro Pro Ser Ala Pro Pro Pro Ser 1580 1589 1598 1607 1616 1625 GGA TCT GGC ACA AGT AAA ATA GAC AAA TAT GCC CGT ATT CTC TTT CCA GTC ACA Gly Ser Gly Thr Ser Lys He Asp Lys Tyr Ala Arg He Leu Phe Pro Val Thr 1634 1643 1652 1661 1670 1679 TTT GGG GCA TTT AAC ATG GTT TAT TGG GTT GTT TAT TTA TCT AAG GAC ACT ATG Phe Gly Ala Phe Asn MET Val Tyr Trp Val Val Tyr Leu Ser Lys Asp Thr MET 1688 1697 1707 > GAG AAA TCA GAA AGT CTA ATG TGA ATTC Glu Lys Ser Glu Ser Leu MET (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 554 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: MET Val Ser Ala Lys Lys Val Pro Ala He Thr Leu Ser Ala Gly Val Ser Phe Ala 1 5 10 15 Leu Leu Arg Phe Leu Cys Leu Ala Val Cys Leu Asn Glu Ser Pro Gly Gin Asn Gin 20 25 30 35 Lys Glu Glu Lys Leu Cys Thr Glu Asn Phe Thr Arg He Leu Asp Ser Leu Leu Asp 40 45 50 55 Gly Tyr Asp Asn Arg Leu Arg Pro Gly Phe Gly Gly Pro Val Thr Glu Val Lys Thr 60 65 70 75 Asp He Tyr Val Thr Ser Phe Gly Pro Val Ser Asp Val Glu Val Glu Tyr Thr MET 80 85 90 95 Asp Val Phe Phe Arg Gin Thr Trp He Asp Lys Arg Leu Lys Tyr Asp Gly Pro He 100 105 110 Glu He Leu Arg Leu Asn Asn MET MET Val Thr Lys Val Trp Thr Pro Asp Thr Phe 115 120 125 130 Phe Arg Asn Gly Lys Lys Ser Val Ser His Asn MET Thr Ala Pro Asn Lys Leu 135 140 145 150 Phe Arg He MET Arg Asn Gly Thr He Leu Tyr Thr MET Arg Leu Thr He Ser 155 160 165 Ala Glu Cys Pro MET Arg Leu Val Asp Phe Pro MET Asp Gly His Ala Cys Pro 170 175 180 185 Val Lys Phe Gly Ser Tyr Ala Tyr Pro Lys Ser Glu MET He Tyr Thr Trp Thr 190 195 200 205 Lys Gly Pro Glu Lys Ser Val Glu Val Pro Lys Glu Ser Ser Ser Leu Val Gin 210 215 220 Tyr Asp Leu He Gly Gin Thr Val Ser Ser Glu Thr He Lys Ser He Thr Gly 225 230 235 240 Glu Tyr He Val MET Thr Val Tyr Phe His Leu Arg Arg Lys MET Gly Tyr Phe 245 250 255 MET He Gin Thr Tyr He Pro Cys He MET Thr Val He Leu Ser Gin Val Ser 260 265 270 275 Phe Trp He Asn Lys Glu Ser Val Pro Ala Arg Thr Val Phe Gly He Thr Thr 280 285 290 295 Val Leu Thr MET Thr Thr Leu Ser He Ser Ala Arg His Ser Leu Pro Lys Val 300 305 310 Ser Tyr Ala Thr Ala MET Asp Trp Phe He Ala Val Cys Phe Ala Phe Val Phe 315 320 325 330 Ser Ala Leu He Glu Phe Ala Ala Val Asn Tyr Phe Thr Asn He Gin MET Glu 335 340 345 Lys Ala Lys Arg Lys Thr Ser Lys Pro Pro Gin Glu Val Pro Ala Ala Pro Val 350 355 360 365 Gin Arg Glu Lys His Pro Glu Ala Pro Leu Gin Asn Thr Asn Ala Asn Leu Asn 370 375 380 385 MET Arg Lys Arg Thr Asn Ala Leu Val His Ser Glu Ser Asp Val Gly Asn Arg 390 395 400 Thr Glu Val Gly Asn His Ser Ser Lys Ser Ser Thr Val Val Gin Glu Ser Ser 405 410 415 420 Lys Gly Thr Pro Arg Ser Tyr Leu Ala Ser Ser Pro Asn Pro Phe Ser Arg Ala 425 430 435 Asn Ala Ala Glu Thr He Ser Ala Ala Arg Ala Leu Pro Ser Ala Ser Pro Thr 440 445 450 455 Ser He Arg Thr Gly Tyr MET Pro Arg Lys Ala Ser Val Gly Ser Ala Ser Thr 460 465 470 475 Arg His Val Phe Gly Ser Arg Leu Gin Arg He Lys Thr Thr Val Asn Thr He 480 485 490 Gly Ala Thr Gly Lys Leu Ser Ala Thr Pro Pro Pro Ser Ala Pro Pro Pro Ser 495 500 505 510 Gly Ser Gly Thr Ser Lys He Asp Lys Tyr Ala Arg He Leu Phe Pro Val Thr 515 520 525 Phe Gly Ala Phe Asn MET Val Tyr Trp Val Val Tyr Leu Ser Lys Asp Thr MET 530 535 540 545 Glu Lys Ser Glu Ser Leu MET 550 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULETYPE:cDNA (xi)SEQUENCEDESCRIPTION:SEQIDNO:9: AGCCCGAATT CCATGGACGT TCTGGGCTGG CTG (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GGTTTCCAAG CTTACTTTGG AGAGGTAGC (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1555 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 47..1405 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 10 20 30 40 49 58 > GAATTCCCCA AGTTTGCGCG GACCCCGTCC CGAGCCCGCC GCGGCC ATG GAC GCG CCC GCC MET Asp Ala Pro Ala 67 76 85 94 103 112 CGG CTG CTG GCC CCG CTC CTG CTC CTC TGC GCG CAG CAG CTC CGC GGC ACC AGA Arg Leu Leu Ala Pro Leu Leu Leu Leu Cys Ala Gin Gin Leu Arg Gly Thr Arg 121 130 139 148 157 166 GCG ATG AAT GAC ATC GGC GAC TAC GTG GGC TCC AAC CTG GAG ATC TCC TGG CTC Ala MET Asn Asp He Gly Asp Tyr Val Gly Ser Asn Leu Glu He Ser Trp Leu 175 184 193 202 211 220 CCC AAC CTG GAC GGG CTG ATA GCC GGT TAC GCC CGC AAC TTC CGG CCT GGC ATC Pro Asn Leu Asp Gly Leu He Ala Gly Tyr Ala Arg Asn Phe Arg Pro Gly He 229 238 247 256 265 274 GGA GGC CCC CCC GTG AAT GTG GCC CTT GCC CTG GAG GTG GCC AGC ATC GAC CAC Gly Gly Pro Pro Val Asn Val Ala Leu Ala Leu Glu Val Ala Ser He Asp His 283 292 301 310 319 328 ATC TCA GAG GCC AAC ATG GAG TAC ACC ATG ACG GTG TTC CTG CAC CAG AGC TGG He Ser Glu Ala Asn MET Glu Tyr Thr MET Thr Val Phe Leu His Gin Ser Trp 337 346 355 364 373 382 CGG GAC AGC AGG CTC TCC TAC AAC CAC ACC AAC GAG ACC CTG GGC CTG GAC AGC Arg Asp Ser Arg Leu Ser Tyr Asn His Thr Asn Glu Thr Leu Gly Leu Asp Ser 391 400 409 418 427 436 CGC TTC GTG GAC AAG CTG TGG CTG CCC GAC ACC TTC ATC GTG AAC GCC AAG TCG Arg Phe Val Asp Lys Leu Trp Leu Pro Asp Thr Phe He Val Asn Ala Lys Ser 445 454 463 472 481 490 GCC TGG TTC CAC GAC GTG ACG GTG GAG AAC AAG CTC ATC CGG CTG CAG CCC GAC Ala Trp Phe His Asp Val Thr Val Glu Asn Lys Leu He Arg Leu Gin Pro Asp 499 508 517 526 535 544 GGG GTG ATC CTG TAC AGC ATC CGA ATC ACC TCC ACT GTG GCC TGC GAC ATG GAC Gly Val He Leu Tyr Ser He Arg He Thr Ser Thr Val Ala Cys Asp MET Asp 553 562 571 580 589 598 CTG GCC AAA TTC CCC ATG GAC GAG CAG GAG TGC ATG CTG GAC CTG GAG AGC TAC Leu Ala Lys Phe Pro MET Asp Glu Gin Glu Cys MET Leu Asp Leu Glu Ser Tyr 607 616 625 634 643 652 GGT TAC TCA TCG GAG GAC ATC GTC TAC TAC TGG TCG GAG AGC CAG GAG CAC ATC Gly Tyr Ser Ser Glu Asp lie Val Tyr Tyr Trp Ser Glu Ser Gin Glu His He 661 670 679 688 697 706 CAC GGG CTG GAC AAG CTG CAG CTG GCG CAG TTC ACC ATC ACC AGC TAC CGC TTC His Gly Leu Asp Lys Leu Gin Leu Ala Gin Phe Thr He Thr Ser Tyr Arg Phe 715 724 733 742 751 760 ACC ACG GAG CTG ATG AAC TTC AAG TCC GCT GGC CAG TTC. CCA CGG CTC AGC CTG Thr Thr Glu Leu MET Asn Phe Lys Ser Ala Gly Gin Phe Pro Arg Leu Ser Leu 769 778 787 796 805 814 CAC TTC CAC CTG CGG AGG AAC CGC GGC GTG TAC ATC ATC CAA TCC TAC ATG CCC His Phe His Leu Arg Arg Asn Arg Gly Val Tyr He He Gin Ser Tyr MET Pro 823 832 841 850 859 868 TCC GTC CTG CTG GTC GCC ATG TCC TGG GTC TCC TTC TGG ATC AGC CAG GCG GCG Ser Val Leu Leu Val Ala MET Ser Trp Val Ser Phe Trp He Ser Gin Ala Ala 877 886 895 904 913 922 GTG CCC GCC AGG GTG TCT CTA GGC ATC ACC ACG GTG CTG ACG ATG ACC ACG CTC Val Pro Ala Arg Val Ser Leu Gly He Thr Thr Val Leu Thr MET Thr Thr Leu 931 940 949 958 967 976 ATG GTC AGT GCC CGC TCC TCC CTG CCA CGG GCA TCA GCC ATC AAG GCA CTG GAC MET Val Ser Ala Arg Ser Ser Leu Pro Arg Ala Ser Ala He Lys Ala Leu Asp 985 994 1003 1012 1021 1030 GTC TAC TTC TGG ATC TGC TAT GTC TTC GTG TTT GCC GCC CTG GTG GAG TAC GCC Val Tyr Phe Trp He Cys Tyr Val Phe Val Phe Ala Ala Leu Val Glu Tyr Ala 1039 1048 1057 1066 1075 1084 TTT GCT CAT TTC AAC GCC GAC TAC AGG AAG AAG CAG AAG GCC AAG GTC AAG GTC Phe Ala His Phe Asn Ala Asp Tyr Arg Lys Lys Gin Lys Ala Lys Val Lys Val 1093 1102 1111 1120 1129 1138 TCC AGG CCG AGG GCA GAG ATG GAC GTG AGG AAC GCC ATT GTC CTC TTC TCC " CTC Ser Arg Pro Arg Ala Glu MET Asp Val Arg Asn Ala He Val Leu Phe Ser Leu 1147 1156 1165 1174 1183 1192 TCT GCT GCC GGC GTC ACG CAG GAG CTG GCC ATC TCC CGC CGG CAG CGC CGC GTC Ser Ala Ala Gly Val Thr Gin Glu Leu Ala He Ser Arg Arg Gin Arg Arg Val 1201 1210 1219 1228 1237 1246 CCG GGG AAC CTG ATG GGC TCC TAC AGG TCG GTG GGG GTG GAG ACA GGG GAG ACG Pro Gly Asn Leu MET Gly Ser Tyr Arg Ser Val Gly Val Glu Thr Gly Glu Thr 1255 1264 1273 1282 1291 1300 AAG AAG GAG GGG GCA GCC CGC TCA GGA GGC CAG GGG GGC ATC CGT GCC CGG CTC Lys Lys Glu Gly Ala Ala Arg Ser Gly Gly Gin Gly Gly He Arg Ala Arg Leu 1309 1318 1327 1336 1345 1354 AGG CCC ATC GAC GCA GAC ACC ATT GAC ATT TAC GCC CGC GCT GTG TTC CCT GCG Arg Pro He Asp Ala Asp Thr He Asp He Tyr Ala Arg Ala Val Phe Pro Ala 1363 1372 1381 1390 1399 1415 GCG TTT GCG GCC GTC AAT GTC ATC TAC TGG GCG GCA TAC GCC ATG TGA GCACAGGACT Ala Phe Ala Ala Val Asn Val He Tyr Trp Ala Ala Tyr Ala MET . 1425 1435 1445 1455 1465 1475 CAGGCCACCC TCGCTTGTCC TGGCGCCCGG CGGCAGCTGC CCAGAAACTT CCTGGGAGAA 1485 1495 1505 1515 1525 1535 AGAGCCCTCG GGCTGCCTTC CCCTCTGCGT GTTTCGAAGT GGGATGACAG TCGGCCACGG 1545 1555 AAAACAAGAG GAAGCCTCGG (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 452 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: MET Asp Ala Pro Ala Arg Leu Leu Ala Pro Leu Leu Leu Leu Cys Ala Gin Gin 1 5 10 15 Leu Arg Gly Thr Arg Ala MET Asn Asp He Gly Asp Tyr Val Gly Ser Asn Leu Glu 20 25 30 35 He Ser Trp Leu Pro Asn Leu Asp Gly Leu He Ala Gly Tyr Ala Arg Asn Phe Arg 40 45 50 55 Pro Gly He Gly Gly Pro Pro Val Asn Val Ala Leu Ala Leu Glu Val Ala Ser He 60 65 70 75 Asp His He Ser Glu Ala Asn MET Glu Tyr Thr MET Thr Val Phe Leu His Gin Ser 80 85 90 Trp Arg Asp Ser Arg Leu Ser Tyr Asn His Thr Asn Glu Thr Leu Gly Leu Asp Ser 95 100 105 110 Arg Phe Val Asp Lys Leu Trp Leu Pro Asp Thr Phe He Val Asn Ala Lys Ser 115 120 125 130 Ala Trp Phe His Asp Val Thr Val Glu Asn Lys Leu He Arg Leu Gin Pro Asp 135 140 145 Gly Val He Leu Tyr Ser He Arg He Thr Ser Thr Val Ala Cys Asp MET Asp 150 155 160 165 Leu Ala Lys Phe Pro MET Asp Glu Gin Glu Cys MET Leu Asp Leu Glu Ser Tyr 170 175 180 185 Gly Tyr Ser Ser Glu Asp He Val Tyr Tyr Trp Ser Glu Ser Gin Glu His He 190 195 200 His Gly Leu Asp Lys Leu Gin Leu Ala Gin Phe Thr He Thr Ser Tyr Arg Phe 205 210 215 220 Thr Thr Glu Leu MET Asn Phe Lys Ser Ala Gly Gin Phe Pro Arg Leu Ser Leu 225 230 235 His Phe His Leu Arg Arg Asn Arg Gly Val Tyr He He Gin Ser Tyr MET Pro 240 245 250 255 Ser Val Leu Leu Val Ala MET Ser Trp Val Ser Phe Trp He Ser Gin Ala Ala 260 265 270 275 Val Pro Ala Arg Val Ser Leu Gly He Thr Thr Val Leu Thr MET Thr Thr Leu 280 285 290 MET Val Ser Ala Arg Ser Ser Leu Pro Arg Ala Ser Ala He Lys Ala Leu Asp 295 300 305 310 Val Tyr Phe Trp He Cys Tyr Val Phe Val Phe Ala Ala Leu Val Glu Tyr Ala 315 320 325 Phe Ala His Phe Asn Ala Asp Tyr Arg Lys Lys Gin Lys Ala Lys Val Lys Val 330 335 340 345 Ser Arg Pro Arg Ala Glu MET Asp Val Arg Asn Ala He Val Leu Phe Ser Leu 350 355 360 365 Ser Ala Ala Gly Val Thr Gin Glu Leu Ala He Ser Arg Arg Gin Arg Arg Val 370 375 380 Pro Gly Asn Leu MET Gly Ser Tyr Arg Ser Val Gly Val Glu Thr Gly Glu Thr 385 390 395 400 Lys Lys Glu Gly Ala Ala Arg Ser Gly Gly Gin Gly Gly He Arg Ala Arg Leu 405 410 415 Arg Pro He Asp Ala Asp Thr He Asp He Tyr Ala Arg Ala Val Phe Pro Ala 420 425 430 435 Ala Phe Ala Ala Val Asn Val He Tyr Trp Ala Ala Tyr Ala MET 440 445 450 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TAATGAGTTT AAACCATAGC TTCTTCCAGT (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CATGATGGAT CCGCCCGCTC AGAC

Claims: CLAIMS: 1. A stably co-transfected eukaryotic cell line capable of expressing a human GABAA receptor, which receptor comprises the alpha-4 receptor subunit, at least one beta receptor subunit and the delta receptor subunit. 2. A stably co-transfected eukaryotic cell line capable of expressing a human GABAA receptor, which receptor comprises the alpha-4 receptor subunit, at least one beta receptor subunit and at least one gamma receptor subunit. 3. A stably co-transfected eukaryotic cell line capable of expressing a human GABAA receptor, which receptor comprises at least one alpha receptor subunit, at least one beta receptor subunit and the delta receptor subunit. 4. A cell line as claimed in any one of claims 1 to 3 wherein the cell line is a rodent fibroblast cell line. 5. A process for the preparation of a eukaryotic cell line capable of expressing a human GABAA receptor, which comprises stably co-transfecting a rodent fibroblast host cell with at least three expression vectors, one such vector harbouring the human cDNA sequence encoding the alpha-4 receptor subunit, another such vector harbouring the human cDNA sequence encoding a beta receptor subunit, and a third such vector harbouring the human cDNA sequence encoding the delta receptor subunit. 6. A process for the preparation of a eukaryotic cell line capable of expressing a human GABAA receptor, which comprises stably co-transfecting a rodent fibroblast host cell with at least three expression vectors, one such vector harbouring the human cDNA sequence encoding the alpha-4 receptor subunit, another such vector harbouring the human cDNA sequence encoding a beta receptor subunit, and a third such vector harbouring the human cDNA sequence encoding a gamma receptor subunit. 7. A process for the preparation of a eukaryotic cell line capable of expressing a human GABAA receptor, which comprises stably co-transfecting a rodent fibroblast host cell with at least three expression vectors, one such vector harbouring the human cDNA sequence encoding an alpha receptor subunit, another such vector harbouring the human cDNA sequence encoding a beta receptor subunit, and a third such vector harbouring the human cDNA sequence encoding the delta receptor subunit. 8. A process as claimed in any one of claims 5 to 7 wherein the eukaryotic cell line is a rodent fibroblast cell line. 9. A DNA molecule encoding the cu subunit of the human GABAA receptor comprising all or a portion of the sequence depicted in Figure 2 herein SEQ. ID. NO.: 7. 10. A DNA molecule encoding the δ subunit of the human GABAA receptor comprising all or a portion of the sequence depicted in Figure 3 herein SEQ. ID. NO.: 10. 11. A recombinant expression vector comprising the nucleotide sequence of the human o GAB AA receptor subunit together with additional sequences capable of directing the synthesis of the said human cu GABAA receptor subunit in cultures of stably co-transfected eukaryotic cells. 12. A recombinant expression vector comprising the nucleotide sequence of the human δ GABAA receptor subunit together with additional sequences capable of directing the synthesis of the said human δ GABAA receptor subunit in cultures of stably co-transfected eukaryotic cells. 13. A protein preparation of human GABAA receptor subunit combinations comprising the human ou GABAA receptor subunit derived from a culture of stably co-transfected eukaryotic cells. 14. A protein preparation of human GABAA receptor subunit combinations comprising the human δ GABAA receptor subunit derived from a culture of stably co-transfected eukaryotic cells. 15. A membrane preparation containing GABAA receptor subunit combinations comprising the human cu GABAA receptor subunit derived from a culture of stably co-transfected eukaryotic cells. 16. A membrane preparation containing GABAA receptor subunit combinations comprising the human δ GABAA receptor subunit derived from a culture of stably co-transfected eukaryotic cells. 17. A preparation as claimed in claim 13 or 14 wherein the subunit combination derived is the cuβ 3 δ subunit combination of the human GABAA receptor. 18. A preparation as claimed in claim 13 wherein the subunit combination derived is the o β3Y2 subunit combination of the human GABAA receptor. 19. A preparation as claimed in claim 13 wherein the subunit combination derived is the c β2γ2 subunit combination of the human GABAA receptor. 20. A preparation as claimed in claim 14 wherein the subunit combination derived is the αββ3δ subunit combination of the human GABAA receptor. 21. A preparation as claimed in claim 15 or 16 wherein the subunit combination derived is the c βaδ subunit combination of the human GABAA receptor. 22. A preparation as claimed in claim 15 wherein the subunit combination derived is the α 4 β3γ 2 subunit combination of the human GABAA receptor. 23. A preparation as claimed in claim 15 wherein the subunit combination derived is the o β2γ2 subunit combination of the human GABAA receptor. 24. A preparation as claimed in claim 16 wherein the subunit combination derived is the αεβsδ subunit combination of the human GABAA receptor. 25. The use of the cell line as claimed in any one of claims 1 to 3, and membrane preparations derived therefrom, in screening for and designing medicaments which act upon the human GABAA receptor.

Inventors: LE BOURDELLES BEATRICE, WHITING PAUL JOHN, LE BOURDELLES, BEATRICE, WHITING, PAUL, JOHN
Applicants: LE BOURDELLES BEATRICE, MERCK SHARP & DOHME, WHITING PAUL JOHN, LE BOURDELLES, BEATRICE, MERCK SHARP & DOHME LIMITED, WHITING, PAUL, JOHN
Application year: 1995
Main classification: C12N15
IPC codes: C07K14, C12N5, C12N15