CA2151236C - Stably transfected cell lines expressing gaba-a receptors - Google Patents

Abstract

The present invention relates to a stably co-transfected eukaryotic cell line capable of expressing a human GABA A receptor, which receptor comprises the .alpha.1.beta.3.gamma.2, .alpha.2.beta.3.gamma.2, .alpha.5.beta.3.gamma.2, .alpha.1.beta.1.gamma.2, .alpha.1.beta.2.gamma.2, .alpha.3.beta.3.gamma.2 or .alpha.6.beta.3.gamma.2 subunit combination; to membrane preparations derived from cultures thereof; and to the use of the cell line in designing and developing GABA A receptor subtype-selective medicaments.

Description

WO 94/13799 ~ 1~ 1 23 6 PCT/GB93/02506 STABLY TRANSFECTED CELL LINES EXPRESSING GABA-A RECEPTORS
This invention concerns a cell line, and in particular relates to a stable cell line capable of = expressing human or animal GABAA receptors. The invention further concerns the cloning of novel cDNA
sequences encoding particular subunits of the human GABAA
receptor. In addition, the invention relates 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 GABAA 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 Q-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 (a, Q, OV, and (5) based on their sequence similarities. To date, six types of a (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 Q(Ymer et al., EMBO J., 1989, 8, 1665), two types of JV (Ymer et al., EMBO J., 1990, 9, 3261; and Shivers et al., Neuron, 1989, 3, 327) and one S subunit (Shivers et al., Neuron, 1989, 3, 327) have been identified.

The differential distribution of many of the subunits has been characterised by in situhybridisation (Sequier et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 7815; Malherbe et al., J. Neurosci., 1990, 10, 2330; and Shivers et al., Neuron, 1989, 3, 327) 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 GABAA 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 al., Nature (London), 1990, 346, 648). This approach has revealed that, in addition to an a and /3 subunit, either r1 or h (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 V3 (Herb et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 1433; Knoflach et al., FEBS Lett., 1991, 293, 191; and Wilson-Shaw et al., 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 a and J' subunits present. Receptors containing a S subunit (i.e. aQS) 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 BZ1 type receptor (a1Q1h) and a BZ2 type receptor (a2Q1h or a3Rd2, Pritchett et al., Nature (London), 1989, 338, 582), as well as two GABAA receptors with a novel pharmacology, a592JV2 (Pritchett and Seeberg, J.
Neurochem., 1990, 54, 1802) and a6/32r2 (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 vivo.
SUMMARY OF INVENTION

The present invention is concerned with the production of permanently transfected cells containing the GABAA receptor, which will be useful for screening for drugs which act on this receptor. The GABAA receptor has previously been expressed in Xenopus oocytes (Sigel et al., Neuron, 1990, 5, 703-711) and in transiently transfected mammalian cells (Pritchett et al., Science, 1989, 245, 1389-1392). However, both of those systems involve transient expression and are unsuitable for screening purposes.

We have now achieved the stable expression of the receptor.

Accordingly, the present invention provides a stably co-transfected eukaryotic cell line capable of expressing a GABAA receptor, which receptor comprises at least one alpha, one beta and one gamma subunit.

This has been achieved by co-transfecting cells with three expression vectors, each harbouring cDNAs encoding for an a, (3 or y 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 GABAA receptor, which comprises stably co-transfecting a eukaryotic host cell with at least three expression vectors, one such vector harbouring the cDNA sequence encoding for an alpha, another such vector harbouring the cDNA sequence encoding for a beta, and a third such vector harbouring the cDNA

-3a-sequence encoding for a gamma GABAA receptor subunit. The stable cell-line which is established expresses an a(3y GABAA receptor. Each receptor thereby expressed, comprising a unique combination of a, P and y subunits, will be referred to hereinafter as a GABAA receptor "subunit combination". Pharmacological and electrophysiological data confirm that the recombinant aRy receptor expressed by the cells of the present invention has the properties expected of a native GABAA
receptor.

Expression of the GABAA 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 mammalian 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 at least one a, one R and one y subunit into the cell line in order to produce the required receptor. Within this limitation, the choice of receptor subunit combination is made according to the type of activity or selectivity which is being screened for. For example, benzodiazepines (designated BZ) represent one class of drugs which act upon the GABAA receptor. The presence of anal subunit is specific for a class of benzodiazepines having the pharmacology designated BZ1i whereas a2 to as define different pharmacological profiles, broadly designated as BZ2. The type of R subunit is not critical in defining the class of benzodiazepine, although a R subunit is required. The y subunit is also important in defining BZ
selectivity. It is likely that differentiation between a subunit selectivity is conferred by the identity of the particular y subunit present.

-4a-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: a1P1Y2; a1P2Y2 ;

2N1y1 ; (Y2N1y2 ; a'2N1y3 ; C13N1y2 ; R'3N173 ; a4N73 ; a4R172 ; a'5P172 ; and a6P1'y2i especially al(31y2i,.

The invention provides a stably co-transfected rodent fibroblast cell line capable of expressing a human GABAA receptor comprising the cx1i6372 subunit combination under the control of an inducible promoter.

The invention further provides a stably co-transfected rodent fibroblast cell line capable of expressing a human GABAA receptor comprising the a293-y2 subunit combination under the control of an inducible promoter.

The invention still further provides a stably co-transfected rodent fibroblast cell line capable of expressing a human GABAA receptor comprising the cx503-y2 subunit combination under the control of an inducible promoter.

The invention still further provides a stably co-transfected rodent fibroblast cell line capable of expressing a human GABAA receptor comprising one of the 01191T28, 192(2, 393'(2, and CY693'Y2 subunit combinations under the control of an inducible promoter.

The invention further provides a membrane preparation containing subunit combinations of the human GABAA receptor derived from a culture of rodent fibroblast cells stably co-transfected with any one of the cX193-y2, the 01293-y2, and the CY593T2 subunit combination.

The invention further provides a membrane preparation containing subunit combinations of the human -5a-GABAA receptor derived from a culture of rodent fibroblast cells stably co-transfected with any one of the O1191'y2S, the Oe192y2, the 0093'Y2r and the Ct693-y2 subunit combination.

The invention further provides a membrane preparation containing a human GABAA receptor consisting of the the CY193"Y23, the a293-Y28 and the Q'593'Y28 subunit combination isolated from stably co-transfected mouse Ltk- fibroblast cells, wherein said cells are capable of expressing a human GABAA receptor comprising a subunit selected from the group consisting of a193y2s, 2a3'Y2s and 5a3'Y2S.

The invention still further provides a membrane preparation containing a human GABAA receptor consisting of the Otl#l'Y2s. 1N2'Y2S, 01503'Y2s, A'Y2S~ or a693'y2s subunit combination isolated from stably co-transfected mouse Ltk- fibroblast cells, wherein said cells are capable of expressing a human GABAA receptor comprising a subunit selected from the group consisting Of CY191'Y2S r 1N210S i 5N3~(2S i a'393Y2S r and a6P3'Y2S =

In a particular embodiment, the present invention provides a stably co-transfected eukaryotic cell line capable of expressing a human GABAA receptor comprising the cx10372 subunit combination.

In a further embodiment, the present invention provides a stably co-transfected eukaryotic cell line capable of expressing a human GABAA receptor comprising the cx2P3y2 subunit combination.

- 5b -In a still further embodiment, the present invention provides a stably co-transfected eukaryotic cell line capable of expressing a human GABAA receptor comprising the a5(33y2 subunit combination.

In yet further embodiments, the present invention provides stably co-transfected eukaryotic cell lines capable of expressing human GABAA receptors comprising the JP172s, 011P272, (43y2 and c43y2 subunit combinations.

The DNAs for the receptor subunits can be obtained from known sources, and are generally obtained as specific nucleotide sequences harboured by a standard cloning vector such as those described, for example, by Maniatis et al. in Molecular Cloning. A Laboratory Manual, Cold Spring Harbor press, New York, 2nd edition, 1989. Preferably the cDNA sequences are derived from the human gene. However, for screening purposes, cDNAs from other species are also suitable, such as bovine or rat DNA. Known sources of GABAA receptor subunit cDNAs are as follows:

albovine ) Schofield et al., Nature, 1987, 328, (3lbovine ) 221-227.

alhuman ) Schofield et al., FEBS Lett., 1989, 244, (3lhuman ) 361-364.

a2 rat Khrestchatisky et al., J. Neurochem., 1991, 56, 1717.

a2 bovine ) Levitan et al., Nature, 1988, 335, a3 bovine ) 76-79.

a4 rat Wisden et al., FEBS Lett., 1991, 289, 227.
a4 bovine Ymer et al., FEBS Lett., 1989, 258, 119-122.
a5 rat Pritchett-and Seeburg, J. Neurochem., 1990, 54, 1802-1804.
a6 rat ) Luddens et al., Nature, 1990, 346, a6 bovine ) 648-651.

(32 bovine ) Ymer et al., EMBO J., 1989, 8, 1665-1670.
(.i 2 rat Q3 bovine /33 rat 93 human Wagstaff et al., Am. J. Hum. Genet., 1991, 49, 330.
jV1 human ) Ymer et al., EMBO J., 1990, 9, 3261-3267.
rat bovine ) h human Pritchett et al., Nature, 1989, 338, 582-585.
bovine Whiting et al., Proc. Natl. Acad.
Sci. USA, 1990, 57, 9966-9970. =
OV3 rat Herb et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 1433; and ~ WO 94/13799 ~ 36 PCT/GB93/02506 Knoflach et al., FEBS Lett., 1991, 293, 191.

= r3 mouse Wilson-Shaw et al., FEBS Lett., 1991, 284, 211.

6 rat Shivers et al., Neuron, 1989, 3, 327.
Certain cDNA sequences encoding various subunits of the human GABAA receptor have hitherto been unavailable. These include in particular the sequences encoding the a2, a3, a5, a6 and 92 subunits, which nucleotide sequences are accordingly novel. We have now ascertained the cDNA sequences of the a2, a3, a5, a6 and 02 subunits of the human GABAA receptor. These nucleotide sequences, together with the deduced amino acid sequences corresponding thereto, are depicted in Figures 2 to 6 of the accompanying drawings. The present invention accordingly provides in several additional aspects DNA molecules encoding the a2, a3, a5, a6 and 02 subunits of the human GABAA receptor comprising all or a portion of the sequences depicted in Figures 2, 3, 4, 5 and 6 respectively, or substantially similar sequences.
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 3; 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 Clonina, 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 a GABAA receptor subunit together with additional sequences capable of directing the synthesis of the said GABAA receptor 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 chosen GABAA 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 ~ WO 94/13799 2151236 PCT/GB93/02506 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 ampicillin 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 GABAA 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 as described in Example 1 herein.
The recombinant expression vector of the present invention is then produced by cloning the GABAA
receptor subunit cDNA into the above precursor vector.
The required receptor subunit cDNA is subcloned from the vector in which it is harboured, and ligated into a restriction enzyme site, e.g. the HindIII 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 in Example 1, step (b) herein. Before this subcloning, it is often advantageous, in order to improve expression, to modify the end of a subunit cDNA
with additional 5' untranslated sequences, for example by modifying the 5' end of the e2L subunit DNA by addition of 5'-untranslated region sequences from the cz1 subunit DNA.

WO 94/13799 . PCT/GB93/02506 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 a given alpha, beta or gamma subunit of the GABAA
receptor, and the remainder of the expression vector depicted therein is derived from the precursor vector pMSGneo and constructed as described in accompanying Example 1, steps (a) and (b).
For each cell line of the present invention, three such vectors will be necessary, one containing an a subunit, one containing a Q subunit, and the third containing a 6v 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 et 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 GABAA receptor subunit combinations, especially human GABAA receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells. The invention also provides ~ WO 94/13799 2151236 PCT/GB93/02506 preparations of membranes containing subunit combinations of the GABAA receptor, especially human GABAA receptor subunit combinations, derived from cultures of stably transfected eukaryotic cells. In particular, the protein preparations and membrane preparations according to the invention will suitably contain the a1g1hL, a193,,K2, a2Q36'2 , a5Q3h, a1Q1hS - a02h, a3Q3r2 or a603h subunit combinations of the human GABAA receptor, and will preferably contain a human GABAA receptor consisting of the a1R1J'2L, a1Q3hS, a2Q3r2S, a59341"2S, alQ106S, a1a2hS, a3,63hS or a03hS subunit combinations. In an especially preferred embodiment, the invention provides cell membranes containing a human GABAA receptor consisting of the a1p1o"2L, alP3~2S- a2Q3Y2S, a5Q3J2S, alQ1hS, a1Q2e2S, a3A3r2S or a6Q342S subunit combinations isolated from stably transfected mouse Ltk- fibroblast 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, Q-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 GABAA 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.

PREPARATION OF ai(31~'2L TRANSFECTED CELLS

a) Construction of eukaryotic expression vector pMSGneo The approx. 2500 base pair HindIII-EcoRI

fragment of the vector pMSG (purchased from Pharmacia Biosystems Limited, Milton Keynes, United Kingdom), containing the gpt structural gene and SV40 polyadenylation signals was replaced by the approx. 2800 base pair HindIII-EcoRI fragment of pSV2neo (Southern, P.J. and Berg, P.J., Molecular and Applied Genetics, 1, 327-341, 1982) containing the neomycin resistance gene Neor and SV40 polyadenylation signals. The EcoRI and HindIII sites were then removed by restriction digesting, blunt ending with klenow polymerase, and religating.
EcoRI and HindIII cloning sites were then inserted at the XhoI and SmaI sites of the polylinker by conventional techniques using EcoRI and HindIII linkers.
b) Cloning of subunit cDNAs into pMSGneo Bovine al and (31 GABAA receptor cDNAs were obtained from the Molecular Neurobiology Unit, MRC
Centre, Hills Road, Cambridge (Scholfield, P. et al.
Nature, 328, 221-227, 1987). Bovine h cDNA was cloned by the method of Whiting, P. et al. (Proc. Natl. Acad.
Sci. USA, 87, 9966-9970, 1990). Bovine al was subcloned from pbGRasense by digestion with EcoRI, blunt ending the DNA with klenow polymerase, addition of HindIII linkers by ligation, digestion with HindIII and ligation into the HindIIl site of pMSGneo. Bovine /31 was subcloned from pbGR(3sense by restriction digestion with EcoRI (partial digestion), klenow polymerase blunt ending, ligation of HindIII linkers, restriction digestion with HindIIl and ligation into HindIIl site of pMSGneo. Before subcloning into pMSGneo, the bovine ~2 cDNA was modified from the published sequence as follows. The 5' untranslated region of the bovine a1 cDNA (bases 60-200 of the published sequence) was added to the 5' end of the published h sequence by amplifying the a1 untranslated region using polymerase chain reaction, and then subcloning the product into the 5' BamHI (site in the polylinker of the BluescriptTM Sk cloning vector;
BluescriptTM vector purchased from Stratagene, San Diego, U.S.A.) HindIIl sites of the h cDNA. The modified JV2 cDNA was then subcloned into pMSGneo by digestion with XbaI (site in the polylinker of the cloning vector), blunt ending with kienow polymerase, ligation of XhoI
linkers, digestion with XhoI (site in the polylinker of the cloning vector), and ligation into XhoI site of pMSGneo.

c) Co-transfection of mouse Ltk cells Ltk cells were obtained from the Salk Institute for Biological Studies, San Diego, California.
Cells were grown at 37 C, 5-8o C02, in Modified Eagles Medium containing penicillin, streptomycin and 10% fetal calf serum. The expression vector harbouring the GABAA
receptor subunit DNAs for co-transfection was prepared by a standard protocol (Chen, C. and Okayama, H., BioTechniques, 6, 632-638, 1988). For co-transfection, Ltk- cells were plated in dishes (approx. 2xl05 cells/dish) and grown overnight. The transfection was performed by calcium phosphate precipitation using a kit (purchased from 5 Prime -> 3 Prime Products, Westchester, Pennsylvania). Co-transfection was performed according to manufacturers' instructions, using 5 g of each subunit DNA construct per 10cm dish of cells. After 2 days in culture the cells were divided 1:8 into culture medium containing lmg/ml neomycin [Geneticin (obtainable from Gibco BRL, Paisley, Scotland, U.K.)]. After a further week the concentration was increased to 1.5mg/ml; and.
then 2mg/mi 1 week after that. Resistant clones of cells were isolated and subcloned using cloning cylinders.
Subclones were analysed using radioligand binding:
subclones were grown in 10cm culture dishes, and when confluent changed into culture medium containing l M
dexamethasone (obtainable from Sigma Chemical Company, Poole, Dorset, United Kingdom). 3-5 days later the cells were harvested, membranes prepared and used for radioligand binding (see Example 2, step (a) below) using the benzodiazepine antagonist 3H Rol5-1788 (obtained from New England Nuclear, Du Pont (U.K.) Ltd, Stevenage, United Kingdom). The clone expressing the highest amount of 3H Ro15-1788 binding was subcloned from a single cell by limiting dilution. The resultant clonal population of cells described below is referred to as population A.

CHARACTERIZATION OF a1Q1J'2L TRANSFECTED CELLS

a) Radioligand bindinct The nature of the recombinant e101e2L GABAA
receptors prepared as described in Example 1 was addressed by characterization of the benzodiazepine (BZ) binding pharmacology, using the BZ antagonist 3H Ro15-1788. For radioligand binding assays, cells which had been induced by culture in dexamethasone containing medium for 3-5 days were scraped off into 50mM Tris, pH7.5, 100mM NaCl in the form of Tris buffered saline (TBS) and pelleted (20,000rpm, Sorvall RC5C centrifuge).
The cell pellet was resuspended in 50mM Tris, pH7.5, homogenised using an ultra-ThurraxTMhomogeniser and then pelleted as above. This was repeated once more, and the cells then resuspended in TBS (0.4m1 per original 10cm dish of cells). Radioligand binding was performed in 0.1m1 final volume TBS, containing 5-15 fmols of 3H Ro15-1788 binding sites. After 1 hour incubation on ice the membranes were harvested onto filters using a BrandelTM
cell harvester, washed with cold TBS, and bound radioactivity determined by scintillation counting. The recombinant a1p1hL receptors bound 3H Ro15-1788 with high affinity (KD 0.4nM), at levels of up to 200fmols/l0cm dish of cells. No binding was seen to either untransfected Ltk- cells, or population A cells which had not been induced by addition of dexamethasone to the culture medium, confirming that the 3H Ro15-1788 was binding to recombinant a1Q1h GABAA receptors. The 3H Ro15-1788 binding was inhibited by flunitrazepam, CL218872, FG8205, /3CCM, zolpidem and Ro15-4513, confirming the BZ pharmacology of the recombinant receptor. Since it is established that only GABAA
receptors containing an a, a Q and aJ' subunit exhibit BZ
binding (Pritchett, D. et al., Nature, 338, 582-585, 1989) these data confirm the nature of the recombinant aiQ1r2 GABAA receptors expressed by population A cells.
b) Electrophysiology The nature of the GABAA receptor expressed by population A cells has been extensively characterised by electrophysiological techniques, using whole cell patch clamp. Only cells induced by culture in the presence of dexamethasone showed responses to GABA. Concentration response curves to GABA gave a log EC50 of 5.2, and a Hill coefficient of 1.9. The response to GABA was potentiated by BZs flunitrazepam and CL218872, by the barbiturate pentobarbitone, and by the steroid alphaxalone. The response to GABA was antagonised by both bicuculline and picrotoxin. All these electrophysiological data confirm that the recombinant GABAA receptor expressed by population A cells has all of the properties expected of a bona fide GABAA receptor.

ISOLATION AND SEQUENCING OF cDNAS ENCODING HUMAN GABAA
RECEPTOR a2, a3, a5, a6 & P2 SUBUNITS

a) cDNA libraries cDNAs were cloned from human foetal brain (a2, a3), hippocampal (a5, P2) and cerebellum (a6) lambda bacteriophage cDNA libraries. All cDNA libraries were constructed in the.lambdaZAP vector, and were purchased from StratageneT"'(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- NTr' filters (Amersham) according to the manufacturer's instructions_ b) Isolation of cDNA encoding human a2 subunit A bovine a2 cDNA (obtained from E. Barnard, Molecular Neurobiology, University of Cambridge, Hills Road, Cambridge; Levitan et al., Nature, 1988, 335, 76) was labelled to high specific activity (>1.109 cpm/ g) with 32P by random priming and used as a probe. Library filters (8 replica filters) were prehybridised for 3-6 hours at 42 C in 5x SSPE (lx SSPE is 0.18M NaCl, 0.O1M
Na3PO4 [pH7.4], 1mM EDTA), 5x Denhardt's solution, 100 g/mi salmon sperm DNA, 0.1% sodium dodecyl sulphate (SDS), 30% formamide. Hybridisation was performed in the same buffer for 18 hours at 42"C, including 0.5-1.106 cpm 32P-labelled probe per ml of hybridisation buffer.
Filters were washed at 55 C in 5x SSPE (2x 15 minutes) and lx SSPE (2x 15 minutes) and exposed to Kodak XAxT"'film for 1-3 days. Positive clones were plaque purified using standard techniques, and the BluescriptTM plasmid (Stratagene) "rescued" according to manufacturer's instructions. cDNA clones were sequenced on both strands by standard techniques using SequenaseTM II enzyme (United States Biochemicals). The nucleotide sequence of the cDNA encoding the human GABAA receptor a2 subunit, together with the deduced amino acid sequence corresponding thereto, is shown in Fig. 2 of the accompanying drawings.

c) Isolation of cDNA encoding human a3 subunit A bovine a3 cDNA (obtained from E. Barnard, Molecular Neurobiology, University of Cambridge, Hills Road, Cambridge; Levitan et al., Nature, 1988, 335, 76) was labelled to high specific activity with 32P by random priming and used as a probe. Library filters were prehybridised for 3-6 hours at 55 C in 5x SSPE, 5x Denhardt's solution, 0.1% SDS, 100 g/mi salmon sperm DNA, and hybridised for 18 hours, 55 C in the same buffer, containing 0.5-ix 106 cpm/ml of 32P-labelled bovine a3 cDNA as probe. Filters were washed and exposed to X-ray film as described above; cDNA clones were rescued and sequenced as described above. The longest a3 cDNA clone was missing in approximately 100 bp of the 5' end of the coding region. This was obtained by PCR using as primers an oligonucleotide "anchor" primer derived from the T7 primer sequence of BluescriptTM vector (5'AGCGCGCGTAATACGACTCACTATAGGGCGAA3') and an oligonucleotide derived from sequence near the 5' end of the truncated a3 cDNA, containing an internal Hpal site (5'CAGCATGAATTGTTAACCTCATTGTA3'). Oligonucleotides were.
synthesised on an Applied BiosystemsTM 380B synthesiser.
PCR was performed as described above, and a 300bp PCR
product obtained which was double digested with Hpal and Kpnl and subcloned into the similarly cut truncated a3 cDNA to yield a full length human a3 cDNA. The cDNA was sequenced on both strands as described above. The nucleotide sequence of the cDNA encoding the human GABAA
receptor a3 subunit, together with the deduced amino acid sequence corresponding thereto, is shown in Fig. 3 of the accompanying drawings.

d) Isolation of cDNA encoding human a5 subunit A rat a5 cDNA obtained by polymerase chain reaction (PCR) was used as a probe to screen the cDNA
library. For PCR, sequences of the oligonucleotide primers were taken from the published a5 sequences (Khrestchatisky et al., Neuron, 1989, 3, 745) and incorporated a Hind III site for subcloning purposes: 5' ATTATTCAAGCTTGCCATGGACAATGGAATGCTC3' (bp114-148);
5'GGTTTCCAGCTTACTTTGGAGAGGTAGC3' (bp1507-1535). PCR and subcloning of the PCR product into BluescriptTM SK-vector (Stratagene) for analysis was performed as described elsewhere (Whiting et al., Proc. Nati. Acad. Sci. USA, 1990, 87, 9966) except that rat brain cDNA was used as template. The rat a5 cDNA was labelled with 32P and used to screen the human hippocampal cDNA library, and positive a5 clones rescued and sequenced as described for a2 above. The nucleotide sequence of the cDNA encoding the human GABAA receptor a5 subunit, together with the deduced amino acid sequence corresponding thereto, is shown in Fig. 4 of the accompanying drawings.

e) Isolation of cDNA encoding human a6 subunit A rat a6 cDNA obtained by PCR was used as a probe to screen the cDNA library. PCR was performed as described above for a5, using oligonucleotide primers derived from the published rat a6 sequence (Luddens et al., Nature, 1990, 346, 648) incorporating an EcoRI site for subcloning purposes: 5'GAGGAAGAATTCAGGAGGGTGACCT3' (bp48-72); 5'GAAAATAACGAATTCCAGTGTCCAGCTTT3' (bp1376-1404)_ The rat a6 cDNA clone isolated by PCR was labelled with 32P and used to screen a human cerebellum cDNA library, as described above for a2. Positive a6 clones were purified,- rescued and sequenced as described above. None of the cDNAs contained a complete coding region. To obtain a full length cDNA 3 clones were joined together using convenient restriction sites. The nucleotide sequence of the cDNA encoding the human GABAA
receptor a6 subunit, together with the deduced amino acid sequence corresponding thereto, is shown in Fig. 5 of the accompanying drawings.

f) Isolation of cDNA encoding human Q2 subunit Human 02 cDNA was isolated using as a probe a short human 02 cDNA obtained by PCR. PCR was performed as described above (except that the human cerebellum cDNA
library was used as template), using oligonucleotide primers derived from the published rat 02 sequence (Ymer et al., EMBO J., 1989, 8, 1665), incorporating EcoRI
sites for subcloning purposes: 5' CAAAAGAATTCAGCTGAGAAAGCTGCTAATGC3' (bp1088-1119); 5' TCAGGCGAATTCTCTTTTGTGCCACATGTCGTTC3' (bp1331-1364). The human (32 clone obtained by PCR was radiolabelled with 32P
and used to screen a human hippocampal cDNA library, as described above for a2. The largest cDNA clone obtained lacked the 5' 500bp of the coding reaion of the A2 subunit. This was obtained by PCR using as primers an oligonucleotide "anchor" primer derived from the T7 primer sequence of the BluescriptTM vector (5' AGCGCGCGTAATACGACTCACTATAGGGCGAA3'), and an oligonucleotide derived from sequence near the 5' end of the truncated 02 cDNA, containing a Kpnl site (5' CATCCAGTGGGTACCTCCTTAGGT3'). PCR was performed as described above, and a 700bp PCR product obtained which was digested with kpnl and subcloned into the truncated cDNA clone (also Kpnl digested) to yield a full length human 02 cDNA. The nucleotide sequence of the cDNA
encoding the human GABAA receptor p2 subunit, together with the deduced amino acid sequence corresponding thereto, is shown in Fig.-6 of the accompanying drawings.

WO 94/13799 PCT/GB93/02506 ~

PREPARATION OF STABLY TRANSFECTED CELLS EXPRESSING
a1Q3J'2S, a2Q3J2S AND a5Q3J'2S SUBUNIT COMBINATIONS OF THE
HUMAN GABAA RECEPTOR

Isolation and sequence of human a2 and a5 cDNAs have been described in Example 3. The sequence of human al cDNA has been published previously by Schofield et al., FEBS Lett., 1989, 244, 361. It differs from the bovine sequence at a single amino acid (trp95 in bovine al; arg in human al). To create a human al cDNA the bovine sequence was converted to the human by site directed mutagenesis of amino acid 95 with the oligonucleotide 5'GCAATGAAAATCCGGACTGGCAT3', using methods described elsewhere (K. Wafford and P. Whiting, FEBS Lett., 1992, 313, 113-117). The sequence of human h has been published previously by Pritchett et al., Nature, 1989, 338, 582. A human h cDNA was isolated by PCR using conditions described elsewhere (Whiting et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 9966-9970), using human hippocampal cDNA library as template and oligonucleotide primers derived from the 5' and 3' untranslated regions of the published ?2 sequence, incorporating a Hind III restriction site:
5'GGGAGGGAAGCTTCTGCAACCAAGAGGC3', 5'ACCACATAGAAGCTTATTTAAGTGGAC3'. Sequencing indicated that the form of h used is the short form, J~12S, lacking the 24 bp insert in the putative cytoplasmic loop region (Whiting et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 9966-9970). The sequence of human 03 has been published by Wagstaff et al., Am. J. Hum. Genet., 1991, 41, 330-337. A human 03 cDNA was isolated by screening a human foetal brain cDNA library (see Example 3) with a short human 93 cDNA probe encoding the putative cytoplasmic loop domain which had been obtained using PCR.

Human al, a2, a5, Q3 and hS cDNAs were subcloned into the eukaryotic expression vector pMSGneo (see Example 1) using standard techniques (cf. Maniatis et al. in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York, 2nd Edition, 1989) and stable cell lines expressing human a1Q3hS, a20342S and a5p3hS GABAA receptors were established as described in Example 1.

PREPARATION OF STABLY TRANSFECTED CELLS EXPRESSING
Q1Q1J'2S1 aiQ2J2S, a3Q3~t2S AND a6Q3,:K2S SUBUNIT
COMBINATIONS OF THE HUMAN GABAA RECEPTOR

Isolation of a3 and a6 cDNAs is as described in Example 3, and isolation of a1, (.33 and hS cDNAs is as described above in Example 4. Human (31 subunit cDNA was isolated by PCR from human brain cDNA as described above.
Oligonucleotide primers used for the PCR were derived from the published human Pl sequence (Schofield et al., FEBS Lett., 1989, 244, 361-364), 5' and 3' untranslated regions incorporating Hind III restriction enzyme sites for subcloning:-5'TAATCAAGCTTAGTAATGTGGACAGTACAAAAT3' and 5'AAATGGAAGCTTTAGAACAGACCTCAGTGTACA3'. Human a1, a3, a6, Qi, P2, P3 and hS cDNAs were subcloned into the eukaryotic expression vector pMSGneo (see Example 1) using standard techniques (cf. Maniatis et al. in Molecular Clonina, A Laboratory Manual, Cold Spring Harbor Press, New York, 2nd Edition, 1989) and stable cell lines expressing human a1Q1d"2S, a1Q2hS- a3Q362S and a6630~'2S GABAA receptors were established as described in Example 1.

SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT:
(A) NAME: Merck Sharp & Dohme Limited (B) STREET: Hertford Road (C) CITY: Hoddesdon (D) STATE: Hertfordshire (E) COUNTRY: England (F) POSTAL CODE (ZIP): EN11 9BU

(ii) TITLE OF INVENTION: Stably transfected cell lines expressing GABA-A receptors (iii) NUMBER OF SEQUENCES: 10 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2310 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 298..1683 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Met Asp Asn GLy Met Phe Ser Gly Phe lie Met ILe Lys Asn Leu Leu Leu Phe Cys lie Ser Met Asn Leu Ser Ser His Phe GLy Phe Ser Gln Met Pro Thr Ser Ser Val Lys Asp GLu Thr Asn Asp Asn Ile Thr ILe Phe Thr Arg Ile Leu Asp Gly Leu Leu Asp GLy Tyr Asp Asn Arg Leu 3 5 Arg Pro GLy Leu Gly Glu Arg Ile Thr GLn Vat Arg Thr Asp Ile Tyr WO 94/13799 2151cl 36 PCT/GB93/02506 0 Vat Thr Ser Phe Gly Pro Val Ser Asp Thr Glu Met GLu Tyr Thr Ile Asp Val Phe Phe Arg Gin Ser Trp Lys Asp Glu Arg Leu Arg Phe Lys Gly Pro Met Gln Arg Leu Pro Leu Asn Asn Leu Leu ALa Ser Lys ILe Trp Thr Pro Asp Thr Phe Phe His Asn Gly Lys Lys Ser ILe Ala His Asn Met Thr Thr Pro Asn Lys Leu Leu Arg Leu Glu Asp Asp Gly Thr Leu Leu Tyr Thr Met Arg Leu Thr Ile Ser Ala GLu Cys Pro Met GLn Leu Glu Asp Phe Pro Met Asp ALa His Ala Cys Pro Leu Lys Phe GLy 3 0 Ser Tyr ALa Tyr Pro Asn Ser GLu Vat Vat Tyr Vat Trp Thr Asn Gly Ser Thr Lys Ser VaL Val Val ALa GLu Asp Gly Ser Arg Leu Asn GLn Tyr His Leu Met Gly Gin Thr Val Gly Thr GLu Asn Ile Ser Thr Ser Thr Gly Glu Tyr Thr Ite Met Thr Ala His Phe His Leu Lys Arg Lys Ile Gty Tyr Phe Vat Ile Gln Thr Tyr Leu Pro Cys Ile Met Thr Val Ile Leu Ser Gin Val Ser Phe Trp Leu Asn Arg Gtu Ser Val Pro Ata Arg Thr Val Phe Gly Val Thr Thr VaL Leu Thr Met Thr Thr Leu Ser Ile Ser Ata Arg Asn Ser Leu Pro Lys Val Ala Tyr Ala Thr Ala Met Asp Trp Phe Ile Ala Val Cys Tyr ALa Phe Val Phe Ser ALa Leu Ite 3 0 GLu Phe Ala Thr Val Asn Tyr Phe Thr Lys Arg Gty Trp Ala Trp Asp Gty Lys Lys Ala Leu GLu Ala Ata Lys Ile Lys Lys Lys Arg GLu Val WO 94/13799 PCT/GB93/02506~

ILe Leu Asn Lys Ser Thr Asn Ala Phe Thr Thr Gly Lys Met Ser His Pro Pro Asn Ile Pro Lys Glu GLn Thr Pro Ala Gly Thr Ser Asn Thr Thr Ser Val Ser Vat Lys Pro Ser Glu GLu Lys Thr Ser Glu Ser Lys Lys Thr Tyr Asn Ser Ite Ser Lys Ite Asp Lys Met Ser Arg Ile Vat Phe Pro Vat Leu Phe Gly Thr Phe Asn Leu Val Tyr Trp Ala Thr Tyr Leu Asn Arg Gtu Pro Val Ile Lys GLy Ala Ala Ser Pro Lys GAGGTTTTGC TCACAGGGAC TCTCCATATG TGAGCACTAT CTTTCAGGAA ATTTTTGCAT

GTTTAATAAT ATGTACAAAT AATATTGCCT TGATGTTTCT ATATGTAACT TCAGATGTTT

CCAAGATGTC CCATTGATAA TTCGAGCAAA CAACTTTCTG GAAAAACAGG ATACGATGAC

TGACACTCAG ATGCCCAGTA TCATACGTTG ATAGTTTACA AACAAGATAC GTATATTTTT

AACTGCTTCA AGTGTTACCT AACAATGTTT TTTATACTTC AAATGTCATT TCATACAAAT

TTTCCCAGTG AATAAATATT TTAGGAAACT CTCCATGATT ATTAGAAGAC CAACTATATT

GCGAGAAACA GAGATCATAA AGAGCACGTT TTCCATTATG AGGAAACTTG GACATTTATG

TACAAAATGA ATTGCCTTTG ATAATTCTTA CTGTTCTGAA ATTAGGAAAG TACTTGCATG

ATCTTACACG AAGAAATAGA ATAGGCAAAC TTTTATGTAG GCAGATTAAT AACAGAAATA

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Asp Asn Gly Met Phe Ser Gly Phe Ile Met Ile Lys Asn Leu Leu Leu Phe Cys ile Ser Met Asn Leu Ser Ser His Phe Gly Phe Ser Gln WO 94/13799 PCT/GB93/02506 ~

Met Pro Thr Ser Ser Val Lys Asp Glu Thr Asn Asp Asn lle Thr Ile Phe Thr Arg Ile Leu Asp Gly Leu Leu Asp GLy Tyr Asp Asn Arg Leu Arg Pro Gly Leu Gly Glu Arg Ile Thr Gln Val Arg Thr Asp Ile Tyr Vat Thr Ser Phe Gly Pro Val Ser Asp Thr GLu Met Glu Tyr Thr Ile Asp Val Phe Phe Arg Gln Ser Trp Lys Asp Glu Arg Leu Arg Phe Lys Gly Pro Met Gln Arg Leu Pro Leu Asn Asn Leu Leu Ala Ser Lys Ile Trp Thr Pro Asp Thr Phe Phe His Asn Gly Lys Lys Ser Ile Ala His Asn Met Thr Thr Pro Asn Lys Leu Leu Arg Leu Glu Asp Asp Gly Thr Leu Leu Tyr Thr Met Arg Leu Thr Ile Ser Ala Glu Cys Pro Met Gln Leu Glu Asp Phe Pro Met Asp ALa His Ala Cys Pro Leu Lys Phe Gly Ser Tyr Ala Tyr Pro Asn Ser Glu Val VaL Tyr Val Trp Thr Asn Gly Ser Thr Lys Ser Val Vat Val Ala GLu Asp Gly Ser Arg Leu Asn Gtn Tyr His Leu Met Gty GLn Thr Val Gly Thr Glu Asn Ile Ser Thr Ser Thr Gly Glu Tyr Thr Ile Met Thr Ata His Phe His Leu Lys Arg Lys Ite Gly Tyr Phe Vat Ile Gln Thr Tyr Leu Pro Cys Ile Met Thr Vat Ite Leu Ser Gtn Val Ser Phe Trp Leu Asn Arg Glu Ser Val Pro Ata Arg Thr Vat Phe Gly Vat Thr Thr Vat Leu Thr Met Thr Thr Leu Ser Ile Ser Ala Arg Asn Ser Leu Pro Lys Vat Ala Tyr Ala Thr Ala Met Asp Trp Phe Ite Ata Val Cys Tyr ALa Phe Val Phe Ser Ala Leu ile Glu Phe Ala Thr Val Asn Tyr Phe Thr Lys Arg Gly Trp Ala Trp Asp 2 5 Gly Lys Lys Ala Leu Glu Ala Ala Lys Ile Lys Lys Lys Arg Gtu Vat Ile Leu Asn Lys Ser Thr Asn Ala Phe Thr Thr Gly Lys Met Ser His Pro Pro Asn ILe Pro Lys Glu Gln Thr Pro Ala Gty Thr Ser Asn Thr Thr Ser Val Ser Vat Lys Pro Ser Glu Glu Lys Thr Ser Glu Ser Lys Lys Thr Tyr Asn Ser Ite Ser Lys Ite Asp Lys Met Ser Arg Ile Vat Phe Pro Val Leu Phe Gly Thr Phe Asn Leu Vat Tyr Trp Ala Thr Tyr Leu Asn Arg Glu Pro Val Ite Lys GLy Ala Ala Ser Pro Lys (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1408 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 27..1385 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Met Ala Ser Ser Leu Pro Trp Leu Cys ILe Ile Leu Trp Leu Glu Asn Ala Leu Gly Lys Leu GLu VaL Gtu Gly =WO 94/13799 ~ 151236 PCT/GB93/02506 Asn Phe Tyr Ser Glu Asn Vat Ser Arg Ile Leu Asp Asn Leu Leu Gtu Gly Tyr Asp Asn Arg Leu Arg Pro Gly Phe Gly Gly Ata Vat Thr Glu Vat Lys Thr Asp Ile Tyr Vat Thr Ser Phe Gly Pro Vat Ser Asp Vat Glu Met Gtu Tyr Thr Met Asp Vat Phe Phe Arg Gln Thr Trp Thr Asp Glu Arg Leu Lys Phe Gly Gty Pro Thr Gtu Ile Leu Ser Leu Asn Asn Leu Met Vat Ser Lys Ile Trp Thr Pro Asp Thr Phe Phe Arg Asn GLy Lys Lys Ser Ite Ata His Asn Met Thr Thr Pro Asn Lys Leu Phe Arg 3 0 Ite Met Gtn Asn Gly Thr Ile Leu Tyr Thr Met Arg Leu Thr lie Asn Ala Asp Cys Pro Met Arg Leu Vat Asn Phe Pro Met Asp Gty His Ala Cys Pro Leu Lys Phe Gly Ser Tyr ALa Tyr Pro Lys Ser Glu Ile ILe Tyr Thr Trp Lys Lys GLy Pro Leu Tyr Ser Vat Glu Val Pro Glu Glu Ser Ser Ser Leu Leu GLn Tyr Asp Leu ILe Gly Gin Thr Vat Ser Ser GLu Thr lie Lys Ser Asn Thr GLy Glu Tyr Vat ILe Met Thr Vat Tyr Phe His Leu Gln Arg Lys Met Gly Tyr Phe Met Ile Gin Ile Tyr Thr Pro Cys Ile Met Thr Vat ILe Leu Ser Gin Vat Ser Phe Trp Ile Asn Lys Glu Ser Vat Pro ALa Arg Thr VaL Leu Gly Ile Thr Thr Vat Leu Thr Met Thr Thr Leu Ser Ile Ser Ala Arg His Ser Leu Pro Lys VaL

Ser Tyr ALa Thr ALa Met Asp Trp Phe ILe Ala Val Cys Phe ALa Phe Vat Phe Ser Ala Leu Ile Glu Phe Ala Ala Vat Asn Tyr Phe Thr Asn Leu Gtn Thr Gln Lys Ala Lys Arg Lys Ala GLn Phe Ala Ata Pro Pro Thr Vat Thr ILe Ser Lys Ala Thr Glu Pro Leu Glu Ala Glu Ile Vat Lou Hio Pro Asp Ser Lys Tyr His Leu Lys Lys Arg ILe Thr Ser Lou Ser Leu Pro ILe Vat Ser Ser Ser Glu Ala Asn Lys VaL Leu Thr Arg Ala Pro ILe Leu GLn Ser Thr Pro Vat Thr Pro Pro Pro Leu Pro Pro Ala Phe GLy Gly Thr Ser Lys Ile Asp Gln Tyr Ser Arg Ile Leu Phe 3 0 Pro Vat Ala Phe Ala Gly Phe Asn Leu Vat Tyr Trp Vat Vat Tyr Leu Ser Lys Asp Thr Met Glu VaL Ser Ser Ser Val GLu WO 94/13799 PCT/GB93/02506 ~

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 453 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Ala Ser Ser Leu Pro Trp Leu Cys Ile ILe Leu Trp Leu Gtu Asn Ala Leu Gly Lys Leu Glu Val Glu Gly Asn Phe Tyr Ser Glu Asn Vat 20 Ser Arg Ile Leu Asp Asn Leu Leu Glu Gly Tyr Asp Asn Arg Leu Arg Pro Gty Phe Gty Gly ALa Vat Thr Glu Val Lys Thr Asp Ile Tyr Val Thr Ser Phe Gly Pro Val Ser Asp Vat Glu Met Glu Tyr Thr Met Asp Vat Phe Phe Arg Gtn Thr Trp Thr Asp Glu Arg Leu Lys Phe Gty Gly Pro Thr Glu Ile Leu Ser Leu Asn Asn Leu Met Val Ser Lys Ile Trp 3 5 Thr Pro Asp Thr Phe Phe Arg Asn Gly Lys Lys Ser Ite Ata His Asn WO 94/13799 2~ ~ 1236 PCT/GB93/02506 Met Thr Thr Pro Asn Lys Leu Phe Arg lie Met Gin Asn Gly Thr Ile Leu Tyr Thr Met Arg Leu Thr Ile Asn Ala Asp Cys Pro Met Arg Leu Val Asn Phe Pro Met Asp Gty His Ala Cys Pro Leu Lys Phe Gly Ser Tyr Ala Tyr Pro Lys Ser Glu lle Ile Tyr Thr Trp Lys Lys Gly Pro Leu Tyr Ser Vat Glu Val Pro Gtu Glu Ser Ser Ser Leu Leu Gln Tyr Asp Leu Ile Gly Gln Thr Vat. Ser Ser Glu Thr lle Lys Ser Asn Thr Gly Glu Tyr Val Ite Met Thr Vat Tyr Phe His Leu Gin Arg Lys Met Gty Tyr Phe Met Ile Gln Ile Tyr Thr Pro Cys lle Met Thr Val Ile Leu Ser Gln Val Ser Phe Trp lie Asn tys Glu Ser VaL Pro Ala Arg Thr Vat Leu Gly Ile Thr Thr Val Leu Thr Met Thr Thr Leu Ser lie Ser Ala Arg His Ser Leu Pro Lys VaL Ser Tyr Ala Thr Ala Met Asp Trp Phe Ile Ala Val Cys Phe Ala Phe Vat Phe Ser Ala Leu Ile Glu Phe Ala Ala Val Asn Tyr Phe Thr Asn Leu Gin Thr Gln Lys Ala Lys Arg Lys Ala Gin Phe Ala Ala Pro Pro Thr Val Thr Ile Ser Lys Ala Thr Glu Pro Leu GLu Ala Glu Ile Val Leu His Pro Asp Ser Lys Tyr His Leu Lys Lys Arg Ile Thr Ser Leu Ser Leu Pro Ile Vat Ser Ser Ser GLu Ala Asn Lys VaL Leu Thr Arg Ala Pro Ile Leu Gin Ser Thr Pro Val Thr Pro Pro Pro Leu Pro Pro Ala Phe Gly Gly Thr Ser Lys Ile Asp Gin Tyr Ser Arg Ile Leu Phe Pro Val Ala Phe Ala Gly Phe Asn Leu Val Tyr Trp Val Vai Tyr Leu Ser Lys Asp Thr Met Glu Val 2 5 Ser Ser Ser Val Glu (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1866 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 225..1646 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Trp Arg Val Arg Lys Arg Gly Tyr Phe Gly lie Trp Ser Phe Pro Leu Ile Ile Ala Ala Val Cys Ala GLn Ser Vat Asn Asp Pro Ser Asn Met Ser Leu Val Lys Glu Thr Val Asp Arg Leu Leu Lys Gly Tyr Asp Ile Arg Leu Arg Pro Asp Phe Gly Gty Pro Pro VaL Ala Vat Gly Met Asn Ile Asp Ile Ala Ser Il=e Asp Met VaL Ser Glu Vat Asn Met Asp Tyr Thr Leu Thr Met Tyr Phe Gln Gtn Ala Trp Arg Asp Lys Arg Leu Ser Tyr Asn Val Ile Pro Leu Asn Leu Thr Leu Asp Asn Arg Val ALa Asp Gln Leu Trp VaL Pro Asp Thr Tyr Phe Leu Asn Asp Lys Lys Ser Phe Val His Gly Val Thr Val Lys Asn Arg Met Ite Arg Leu His Pro Asp Gty Thr Vat Leu Tyr Gly Leu Arg Ile Thr Thr Thr Ala ALa Cys Met Met Asp Leu Arg Arg Tyr Pro Leu Asp Gtu Gtn Asn Cys Thr Leu Gtu Ile Glu Ser Tyr GLy Tyr Thr Thr Asp Asp ILe GLu Phe Tyr Trp Arg Gly Asp Asp Asn Ala Val Thr Gly Val Thr Lys Ite Glu Leu Pro Gln Phe Ser ILe 3 5 Val Asp Tyr Lys Leu Ile Thr Lys Lys Val Vat Phe Ser Thr Gly Ser Tyr Pro Arg Leu Ser Leu Ser Phe Lys Leu Lys Arg Asn Ile GLy Tyr Phe lle Leu Gln Thr Tyr Met Pro Ser Ite Leu Ile Thr lle Leu Ser Trp Val Ser Phe Trp Ile Asn Tyr Asp ALa Ser Ala Ala Arg Vat Ala Leu GLy Ile Thr Thr VaL Leu Thr Met Thr Thr lle Asn Thr His Leu CGG GAA ACT CTC CCT AAA ATC CCC TAT GiG AAG GCC ATT GAC ATG TAC 1148 Arg Glu Thr Leu Pro Lys Ile Pro Tyr Val Lys Ala lle Asp Met Tyr Leu Met Gly Cys Phe Val Phe VaL Phe Met Ala Leu Leu Glu Tyr Ala CTA GTC AAC TAC ATC TTC TTT GGG AGG.GGG CCC CAA CGC CAA AAG AAA 1244 Leu Val Asn Tyr ILe Phe Phe Gly Arg Gly Pro Gln Arg Gln Lys Lys Ala Ata GLu Lys Ala Ala Ser Ala Asn Asn Gtu Lys Met Arg Leu Asp VaL Asn Lys Met Asp Pro His GLu Asn Ile Leu Leu Ser Thr Leu GLu Ile Lys Asn Glu Met Ala Thr Ser Glu Ala Val Met Gty Leu Gly Asp Pro Arg Ser Thr Met Leu Ala Tyr Asp Ala Ser Ser Ile Gln Tyr Arg Lys Ala Gly Leu Pro Arg His Ser Phe Gly Arg Asn Ala Leu Glu Arg His Vat Ala Gln Lys Lys Ser Arg Leu Arg Arg Arg Ala Ser GLn Leu Lys Ite Thr Ile Pro Asp Leu Thr Asp Val Asn Ala Ile Asp Arg Trp Ser Arg Ite Phe Phe Pro Vat Val Phe Ser Phe Phe Asn lle Val Tyr Trp Leu Tyr Tyr Val Asn CTAGATTCCT CCTCAAACCA GTTGTACAGC CTGATGTAGG ACTTGGAAAA CACATCAATC

CAGGACAAAA GTGACGCTAA AATACCTTAG TTGCTGGCCT ATCCTGTGGT CCATTTCATA

(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 474 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Trp Arg VaL Arg Lys Arg Gly Tyr Phe Gly ILe Trp Ser Phe Pro Leu ILe ILe ALa Ala VaL Cys Ala Gln Ser Val Asn Asp Pro Ser Asn Met Ser Leu Val Lys Glu Thr Val Asp Arg Leu Leu Lys Gly Tyr Asp ILe Arg Leu Arg Pro Asp Phe Gly Gly Pro Pro VaL Ala Val Gly Met Asn Ile Asp ILe Ala Ser ILe Asp Met Vat Ser Glu Val Asn Met Asp Tyr Thr Leu Thr Met Tyr Phe Gln Gln Ala Trp Arg Asp Lys Arg Leu Ser Tyr Asn Vat Ile Pro Leu Asn Leu Thr Leu Asp Asn Arg VaL ALa Asp Gin Leu Trp Val Pro Asp Thr Tyr Phe Leu Asn Asp Lys Lys Ser Phe Val His Gly Vat Thr Vat Lys Asn Arg Met lie Arg Leu His Pro Asp Gly Thr Val Leu Tyr Gly Leu Arg Ile Thr Thr Thr Ala Ala Cys Met Met Asp Leu Arg Arg Tyr Pro Leu Asp Gtu Gin Asn Cys Thr Leu GLu Ite Glu Ser Tyr GLy Tyr Thr Thr Asp Asp Ile Gtu Phe Tyr Trp Arg Gly Asp Asp Asn Ala Val Thr Gly Vat Thr Lys Ile Glu Leu Pro GLn Phe Ser Ile Vat Asp Tyr Lys Leu ILe Thr Lys Lys Vat Vat Phe Ser Thr Gly Ser Tyr Pro Arg Leu Ser Leu Ser Phe Lys Leu Lys Arg Asn ILe GLy Tyr Phe lie Leu Gin Thr Tyr Met Pro Ser Ile Leu lie Thr Ite Leu Ser Trp Vat Ser Phe Trp Ile Asn Tyr Asp Ala Ser ALa ALa Arg Val Ala Leu Gly ILe Thr Thr Val Leu Thr Met Thr Thr ILe Asn Thr His Leu Arg Gtu Thr Leu Pro Lys ILe Pro Tyr VaL Lys Ala 3 5 ILe Asp Met Tyr Leu Met Gly Cys Phe Vat Phe Vat Phe Met Ala Leu WO 94/13799 r PCT/GB93/02506 Leu Glu Tyr Ala Leu Val Asn Tyr Ile Phe Phe Gly Arg Gly Pro Gin Arg Gln Lys Lys Ala Ala Glu Lys Ala Ala Ser Ala Asn Asn Glu Lys Met Arg Leu Asp Val Asn Lys Met Asp Pro His Glu Asn Ile Leu Leu Ser Thr Leu Glu Ile Lys Asn Glu Met Ala Thr Ser Glu Ala Vat Met Gly Leu Gty Asp Pro Arg Ser Thr Met Leu Ala Tyr Asp Ala Ser Ser Ile Gln Tyr Arg Lys Ala Gly Leu Pro Arg His Ser Phe Gly Arg Asn Ala Leu Glu Arg His Val Ala Gln Lys Lys Ser Arg Leu Arg Arg Arg Ala Ser Gln Leu Lys Ite Thr Ile Pro Asp Leu Thr Asp Val Asn Ala 2 5 Ile Asp Arg Trp Ser Arg Ile Phe Phe Pro Val Val Phe Ser Phe Phe Asn Ile VaL Tyr Trp Leu Tyr Tyr Val Asn (2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2189 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 214..1566 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Met Lys Thr Lys Leu Asn lie Tyr Asn lie Glu Phe Leu Leu Phe Val Phe Leu Val Trp Asp Pro Ala Arg Leu Val Leu Ala Asn Ile GLn Glu Asp Glu Ata Lys Asn Asn Ile Thr Ite Phe Thr Arg Ile Leu Asp Arg Leu Leu Asp Gly Tyr Asp Asn 3 5 Arg Leu Arg Pro Gly Leu Gly Asp Ser Ile.Thr GLu Val Phe Thr Asn Ile Tyr Vat Thr Ser Phe Gly Pro Vat Ser Asp Thr Asp Met Glu Tyr Thr Ile Asp Vat Phe Phe Arg Gln Lys Trp Lys Asp Glu Arg Leu Lys Phe Lys Gly Pro Met Asn Ile Leu Arg Leu Asn Asn Leu Met Ala Ser Lys Ile Trp Thr Pro Asp Thr Phe Phe His Asn Gly Lys Lys Ser Vat Ala His Asn Met Thr Met Pro Asn Lys Leu Leu Arg Ile Gln Asp Asp Gly Thr Leu Leu Tyr Thr Met Arg Leu Thr Val Gln Ala GLu Cys Pro Met His Leu Gtu Asp Phe Pro Met Asp Ala His Ser Cys Pro Leu Lys Phe Gly Ser Tyr Ala Tyr Thr Thr Ser Glu Vat Thr Tyr Ile Trp Thr Tyr Asn Ala Ser Asp Ser VaL Gln Vat Ata Pro Asp GLy Ser Arg Leu Asn Gin Tyr Asp Leu Leu GLy Gin Ser Ile Gly Lys Glu Thr ILe Lys Ser Ser Thr Gly Glu Tyr Thr Val Met Thr Ala His Phe His Leu Lys Arg Lys Ile Gty Tyr Phe Vat Ile GLn Thr Tyr Leu Pro Cys Ile Met Thr Val Ile Leu Ser Gtn Val Ser Phe Trp Leu Asn Arg Glu Ser Vat CCT GCA AGA ACT GTG TTT GGA GTA ACA ACT GTC CTA ACA A;G ACA ACT 1098 Pro Ala Arg Thr Vat Phe Gly Vat Thr Thr VaL Leu Thr Met Thr Thr Leu Ser Ile Ser Ala Arg Asn Ser Leu Pro Lys Vat Ala Tyr Ala Thr Ala Met Asp Trp Phe Ile ALa VaL Cys Tyr Ala Phe VaL Phe Ser Ala 3 0 Leu Ile Glu Phe Ala Thr Val Asn Tyr Phe Thr Lys Arg Gly Trp Thr Trp Asp Gly Lys Ser Vat Val Asn Asp Lys Lys Lys Glu Lys Ala Ser Vat Met Ile Gln Asn Asn Ala Tyr Ala Val Ala Vat Ala Asn Tyr Ala Pro Asn Leu Ser Lys Asp Pro Val Leu Ser Thr Ile Ser Lys Ser Ala Thr Thr Pro Glu Pro Asn Lys Lys Pro Glu Asn Lys Pro Ala Glu Ala Lys Lys Thr Phe Asn Ser Vat Ser Lys Ile Asp Arg Met Ser Arg ile Val Phe Pro Val Leu Phe Gly Thr Phe Asn Leu Val Tyr Trp Ala Thr Tyr Leu Asn Arg Glu Pro Val Leu Gly VaL Ser Pro CTGACTAATA ACTGCTAATT TGTGATCCAA CATGTACAGT ATGTATATAG TGACATAGCT

TACCAGTAGA CCTTTAATGG AGACATGCAT TTGCTAACTC ATGGAACTGC AGACAGAAAG

CACTCCATGC GAAAACAGCC ATTGCCTTTT TTAAAGATTT ACCCTAGGAC CTGATTTAAA

GTGAATTTCA AGTGACCTGA TTAATTTCCT ATTCTTCCAA ATGAGATGAA AATGGGGATC

2t5t236 - 48 -CTGTACAACC CTTTGTGGAC CCTTTTGGTT TAGCTCTTAA GTAGGGGTAT TTTCTACTGT

TGCTTAATTA TGATGGAAGA TAACATTGTC ATTCCTAGAT GAATCCTTTG AAGTAACAAA

CATTGTATCT GACATCAGCT CTGTTCATGA GTGCTCAGAG TCCCTGCTAA TGTAATTGGA

AGCTTGGTAC ACATAAGAAA AACTAGAGAT TTGAAATCTA GCTATGAATT ACTCTATATA

GTATCTATAG CCATGTACAT ATTACAGCAT GACAAGCTCG AAATAATTAT GAGTCAGCCC

(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 451 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Lys Thr Lys Leu Asn Ile Tyr Asn ile Glu Phe Leu Leu Phe VaL

3 5 Phe Leu Val Trp Asp Pro Ala Arg Leu Vat Leu Ala Asn Ile GLn Glu Asp Glu Ala Lys Asn Asn !le Thr Ile Phe Thr Arg Ile Leu Asp Arg Leu Leu Asp Gly Tyr Asp Asn Arg Leu Arg Pro Gly Leu Gty Asp Ser Ile Thr Gtu Val Phe Thr Asn Ile Tyr Vat Thr Ser Phe GLy Pro Vat Ser Asp Thr Asp Met Glu Tyr Thr Ile Asp Vat Phe Phe Arg Gtn Lys Trp Lys Asp Glu Arg Leu Lys Phe Lys Gly Pro Met Asn !le Leu Arg Leu Asn Asn Leu Met Ala Ser Lys Ile Trp Thr Pro Asp Thr Phe Phe His Asn Gly Lys Lys Ser Vat Ala His Asn Met Thr Met Pro Asn Lys Leu Leu Arg ILe Gln Asp Asp Gly Thr Leu Leu Tyr Thr Met Arg Leu Thr Vat Gin Ala Glu Cys Pro Met His Leu Glu Asp Phe Pro Met Asp Ata His Ser Cys Pro Leu Lys Phe Gly Ser Tyr Ala Tyr Thr Thr Ser Glu Vat Thr Tyr Ile Trp Thr Tyr Asn Ala Ser Asp Ser Vat Gln Vat Ala Pro Asp Gly Ser Arg Leu Asn Gln Tyr Asp Leu Leu Gly Gln Ser ILe Gly Lys Glu Thr Ile Lys Ser Ser Thr Gly GLu Tyr Thr Vat Met Thr Ala His Phe His Leu Lys Arg Lys lie Gly Tyr Phe VaL Ile GLn Thr Tyr Leu Pro Cys ILe Met Thr VaL lie Leu Ser Gln Vat Ser Phe Trp Leu Asn Arg Glu Ser VaL Pro Ala Arg Thr Vat Phe Gty Vat Thr Thr Val Leu Thr Met Thr Thr Leu Ser lle Ser Ala Arg Asn Ser Leu Pro Lys Vat Ala Tyr Ala Thr Ala Met Asp Trp Phe Ile Ala VaL Cys Tyr Ala Phe VaL Phe Ser Ala Leu ILe Glu Phe Ala Thr Val Asn Tyr Phe Thr Lys Arg GLy Trp Thr Trp Asp Gty Lys Ser VaL VaL Asn Asp 2 5 Lys Lys Lys Glu Lys Ala Ser Vat Met ILe GLn Asn Asn Ala Tyr Ata Vat Ala VaL Ala Asn Tyr Ala Pro Asn Leu Ser Lys Asp Pro VaL Leu Ser Thr Ile Ser Lys Ser Ala Thr Thr Pro Glu Pro Asn Lys Lys Pro GLu Asn Lys Pro Ala Glu Ala Lys Lys Thr Phe Asn Ser Vat Ser Lys Ile Asp Arg Met Ser Arg Ile Vat Phe Pro Vat Leu Phe Gly Thr Phe Asn Leu Vat Tyr Trp Ala Thr Tyr Leu Asn Arg Gtu Pro Val Leu Gty Val Ser Pro (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1638 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 87..1562 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Met Ite ILe Thr Gln Thr Ser His Cys Tyr Met Thr Ser Leu Gly Ile Leu Phe Leu Ile Asn Ile Leu Pro Gly Thr Thr Gly Gin Gly Glu Ser Arg Arg Gln Glu Pro Gly Asp Phe Val Lys Gln Asp Ile Giy Gly Leu Ser Pro Lys His Ala Pto Asp Ile Pro Asp Asp Ser Thr Asp Asn Ile Thr Ile Phe Thr Arg Ile Leu Asp Arg Leu Leu Asp GLy Tyr Asp Asn Arg Leu Arg Pro Gly Leu Gly Asp Ala Val Thr Glu Val Lys Thr Asp ILe Tyr Val Thr Ser Phe Gly Pro Val Ser Asp Thr Asp Met Glu Tyr Thr Ite Asp Vat Phe Phe Arg Gln Thr Trp His Asp Glu Arg Leu Lys Phe Asp Gly Pro Met Lys Ile Leu Pro Leu Asn Asn Leu Leu Ala Ser Lys ILe Trp Thr Pro Asp Thr Phe Phe His Asn Gly Lys Lys Ser Val Ala His Asn Met Thr Thr Pro Asn Lys WO 94/13799 f v ~ PCT/GB93/02506 Leu Leu Arg Leu Val Asp Asn Gly Thr Leu Leu Tyr Thr Met Arg Leu Thr Ile His Ata Gtu Cys Pro Met His Leu Glu Asp Phe Pro Met Asp VaL His Ala Cys Pro Leu Lys Phe Gly Ser Tyr Ala Tyr Thr Thr Ala Glu Val Vat Tyr Ser Trp Thr Leu Gly Lys Asn Lys Ser Val Glu Val Ala Gin Asp Gly Ser Arg Leu Asn Gln Tyr Asp Leu Leu Gly His Vat Vat Gly Thr Glu Ile Ile Arg Ser Ser Thr Gly Glu Tyr Vat Vat Met Thr Thr His Phe His Leu Lys Arg Lys Ile Gly Tyr Phe Val ILe Gln 3 0 Thr Tyr Leu Pro Cys Ile Met Thr Val Ite Leu Ser GLn Vat Ser Phe Trp Leu Asn Arg GLu Ser Val Pro Ala Arg Thr Val Phe Gty Val Thr Thr Val Leu Thr Met Thr Thr Leu Ser Ile Ser Ala Arg Asn Ser Leu Pro Lys Val Ala Tyr Ala Thr Ala Met Asp Trp Phe Ile Ala Val Cys Tyr Ala Phe Vat Phe Ser Ala Leu Ile Glu Phe Ala Thr Val Asn Tyr Phe Thr Lys Arg Ser Trp Ala Trp Glu Gly Lys Lys Val Pro Glu Ala Leu Glu Met Lys Lys Lys Thr Pro Ala Ala Pro Ala Lys Lys Thr Ser Thr Thr Phe Asn Ite Vat GLy Thr Thr Tyr Pro ile Asn Leu Ala Lys Asp Thr Glu Phe Ser Thr Ile Ser Lys Gly Ala Ala Pro Ser Ala Ser 3 0 Ser Thr Pro Thr Ile Ile Ala Ser Pro Lys Ala Thr Tyr Val Gln Asp Ser Pro Thr Glu Thr Lys Thr Tyr Asn Ser Val Ser Lys Val Asp Lys ]le Ser Arg Ile Ile Phe Pro Val Leu Phe Ala Ile Phe Asn Leu VaL

Tyr Trp Ala Thr Tyr Val Asn Arg Glu Ser Ala Ile Lys Gly Met Ile Arg Lys Gin (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 492 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Ile Ite Thr Gin Thr Ser His Cys Tyr Met Thr Ser Leu Gly Ite Leu Phe Leu Ile Asn Ile Leu Pro Gly Thr Thr Gly GLn Gly Glu Ser Arg Arg Gin Glu Pro Gly Asp Phe Vat Lys Gin Asp Ile Gly Gly Leu Ser Pro Lys His Ala Pro Asp Ile Pro Asp Asp Ser Thr Asp Asn ILe Thr lie Phe Thr Arg Ile Leu Asp Arg Leu Leu Asp Gly Tyr Asp Asn Arg Leu Arg Pro Gly Leu Gly Asp Ala Vat Thr Giu Val Lys Thr Asp Ile Tyr Val Thr Ser Phe Gty Pro Val Ser Asp Thr Asp Met Glu Tyr Thr Ile Asp Val Phe Phe Arg Gtn Thr Trp His Asp Glu Arg Leu Lys Phe Asp Gly Pro Met Lys ILe Leu Pro Leu Asn Asn Leu Leu Ala Ser Lys Ile Trp Thr Pro Asp Thr Phe Phe His Asn Gly Lys Lys Ser Vat Ala His Asn Met Thr Thr Pro Asn Lys Leu Leu Arg Leu Vat Asp Asn Gly Thr Leu Leu Tyr Thr Met Arg Leu Thr Ile His Ala Glu Cys Pro Met His Leu Glu Asp Phe Pro Met Asp Val His Ala Cys Pro Leu Lys Phe Gly Ser Tyr Ala Tyr Thr Thr Ala Glu Vat Vat Tyr Ser Trp Thr Leu Gly Lys Asn Lys Ser Val Glu Val Ala Gtn Asp Gly Ser Arg Leu Asn Gln Tyr Asp Leu Leu Gly His Val Vat Gly Thr Glu Ile lle Arg Ser Ser Thr Gly Gtu Tyr Val Val Met Thr Thr His Phe His Leu Lys Arg Lys Ile Gly Tyr Phe Val Ile Gtn Thr Tyr Leu Pro Cys Ile Met Thr Val Ite Leu Ser Gln Val Ser Phe Trp Leu Asn Arg Glu Ser Vat Pro Ala Arg Thr Vat Phe Gty Vat Thr Thr Val Leu Thr Met Thr Thr Leu Ser Ile Ser Ala Arg Asn Ser Leu Pro Lys Val Ala Tyr ALa Thr 325 330 33:

Ala Met Asp Trp Phe Ile Ala Vat Cys Tyr Ala Phe Vat Phe Ser Ala Leu Ile Gtu Phe Ala Thr Vat Asn Tyr Phe Thr Lys Arg Ser Trp Ala Trp Glu Gty Lys Lys Vat Pro Gtu Ala Leu Glu Met Lys Lys Lys Thr Pro Ala Ala Pro Ata Lys Lys Thr Ser Thr Thr Phe Asn Ile Val Gly Thr Thr Tyr Pro Ile Asn Leu Ala Lys Asp Thr Glu Phe Ser Thr Ile Ser Lys Gly Ala Ala Pro Ser Ala Ser Ser Thr Pro Thr Ite Ite Ala Ser Pro Lys Ala Thr Tyr Val Gln Asp Ser Pro Thr Glu Thr Lys Thr Tyr Asn Ser Val Ser Lys Val Asp Lys Ile Ser Arg Ile Ile Phe Pro Val Leu Phe Ala Ite Phe Asn Leu Val Tyr Trp Ala Thr Tyr Vat Asn Arg Gtu Ser Ala Ile Lys Gty Met Ile Arg Lys Gin

Claims (22)

CLAIMS:

1. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABA A receptor comprising the .alpha.1.beta.3.gamma.2 subunit combination under the control of an inducible promoter.

2. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABA A receptor comprising the .alpha.2.beta.3.gamma.2 subunit combination under the control of an inducible promoter.

3. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABA A receptor comprising the .alpha.5.beta.3.gamma.2 subunit combination under the control of an inducible promoter.

4. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABA A receptor comprising the .alpha.1.beta.1.gamma.2s subunit combination under the control of an inducible promoter.

5. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABA A receptor comprising the .alpha.1.beta.2.gamma.2 subunit combination under the control of an inducible promoter.

6. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABA A receptor comprising the .alpha.3.beta.3.gamma.2 subunit combination under the control of an inducible promoter.

7. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABAA receptor comprising the a61Q3ry2 subunit combination under the control of an inducible promoter.

8. A membrane preparation containing subunit combinations of the human GABAA receptor derived from a culture of the stably co-transfected rodent fibroblast cells as claimed in any one of claims 1 to 3.

9. A membrane preparation containing subunit combinations of the human GABAA receptor derived from a culture of the stably co-transfected rodent fibroblast cells as claimed in any one of claims 4 to 7.

10. A membrane preparation containing a human GABAA

receptor consisting of the a1183'Y2s, 203Y2s or a593y2S
subunit combination isolated from stably co-transfected mouse Ltk- fibroblast cells, wherein said cells are capable of expressing a human GABAA receptor comprising a subunit selected from the group consisting of a1Q3T2s, a203'Y2s and GY5)33Y2S =

11. A membrane preparation containing a human GABAA

receptor consisting of the CY101'Y2s, 102-Y2S, a303Y2S or 693-Y2S subunit combination isolated from stably co-transfected mouse Ltk- fibroblast cells, wherein said cells are capable of expressing a human GABAA receptor comprising a subunit selected from the group consisting of G1191'Y2S, 01192'Y2S, 393Y2s and 01603'Y2s =

12. The use of the cell line as claimed in any one of claims 1 to 3, or membrane preparations derived therefrom, in screening for and designing medicaments which act upon the human GABA A receptor.

13. The use of the cell line as claimed in any one of claims 4 to 7, and membrane preparations derived therefrom, in screening for and designing medicaments which act upon the human GABAA receptor.

14. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABA A receptor comprising the .alpha.1.beta.3.gamma.2s subunit combination under the control of an inducible promoter.

15. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABA A receptor comprising the .alpha.2.beta.3.gamma.2S subunit combination under the control of an inducible promoter.

16. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABAA receptor comprising the .alpha.5.beta.3.gamma.2s subunit combination under the control of an inducible promoter.

17. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABAA receptor comprising the .alpha.1.beta.2.gamma.2s subunit combination under the control of an inducible promoter.

18. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABAA receptor comprising the .alpha.3.beta.3.gamma.2S subunit combination under the control of an inducible promoter.

19. A stably co-transfected rodent fibroblast cell line capable of expressing a human GABA A receptor comprising the .alpha.6.beta.3.gamma.2S subunit combination under the control of an inducible promoter.

20. A membrane preparation containing subunit combinations of the human GABA A receptor derived from a culture of the stably co-transfected rodent fibroblast cells as claimed in any one of claims 14 to 19.

21. A preparation as claimed in claim 20 containing a human GABA A receptor consisting of the .alpha.1.beta.3.gamma.2s, .alpha.2.beta.3.gamma.2s, .alpha.5.beta.3.gamma.2s, .alpha.1.beta.3.gamma.2s, .alpha.6.beta.3.gamma.2s or .alpha.6.beta.3.gamma.2s subunit combination isolated from stably co-transfected mouse Ltk- fibroblast cells, wherein said cells are capable of expressing a human GABA A receptor comprising a subunit selected from the group consisting of .alpha.1.beta.3.gamma.2s, .alpha.5.beta.3.gamma.2s, .alpha.5.beta.3.gamma.2S, .alpha.1.beta.2.gamma.2s, .alpha.3.beta.3.gamma.2s and .alpha.6.beta.3.gamma.2s

22. A use of the cell line as claimed in any one of claims 14 to 19, or membrane preparations derived therefrom, in screening for and designing medicaments which act upon the human GABA A receptor.