WO2001090141A2

WO2001090141A2 - Gb1c isoform and nucleotides

Info

Publication number: WO2001090141A2
Application number: PCT/CA2001/000768
Authority: WO
Inventors: Gordon Yiu Kon Ng
Original assignee: Merck Frosst Canada & Co.
Priority date: 2000-05-25
Filing date: 2001-05-24
Publication date: 2001-11-29
Also published as: WO2001090141A3

Abstract

A new human isoform of the gamma-aminobutyric acid receptor gb1c, termed Gb1c, is described as well as nucleic acids encoding same and various assays employing said receptor. The novel Gb1c cDNA encodes a protein of 899 amino acids differering from the previously noted gb1c sequence (Genbank Accession No. AJ012187) by a single amino acid substitution (a glutamine in place of a histidine at position 21). This single amino acid substitution affords the receptor higher affinity for GABAB-selective antagonists. Accordingly, the disclosed Gb1c receptor can be used to differentially screen for GABAB receptor modulators such as agonists or antagonists for the treatment of various CNS disorders, drug addiction, neuropathic pain and gastroesophageal reflux and incontinence.

Description

TITLE OF THE INVENTION

NOVEL GBIC ISOFORM AND NUCLEOTIDES ENCODING SAME

FIELD OF THE INVENTION This invention relates to a novel receptor, designated Gblc, to nucleotides encoding same, and to assays employing said receptor.

BACKGROUND OF THE INVENTION

Gamma aminobutyric acid (GABA), an endogenous neurotransmitter in the central and peripheral nervous systems, is responsible for mediating synaptic transmission through two ligand-gated channels, GABAA and GAB Ac receptors, and a third receptor sub-type, GABAB, which acts through G proteins to regulate potassium and calcium channels. Specifically, GABAB receptor activation increases

K+ or decreases Ca++ conductance and also inhibits or potentiates stimulated adenylyl cyclase activity. The expression of GABAB receptors is widely distributed in the mammalian brain (e.g., frontal cortex, cerebellar molecular layer, interpeduncular nucleus) and has been observed in many peripheral organs as well. Two GABAB receptors, GABAβRla and GABAβRlb, have been identified that bind GABAB receptor antagonists in transfected cells; Kaupmann et al., 1997 Nature 386:239-246. More recently, a third GABAB receptor isoform, gblc, was reported to GenBank (Accession No. AJ012187). These receptors are extremely valuable in identifying modulators of the GABAB receptor, such as GABAβ agonists and antagonists.

Pharmacological and physiological evidence indicates that a large number of GABAB receptors remain to be cloned. Accordingly, this field continues to be of intense interest. Modified or variant receptors can differ significantly in their functionality. Even a single amino acid change in G protein-coupled receptors can lead to large differences in the receptor's ability to bind ligand (agonist/antagonist) and to couple functionally to signal transduction pathways. For example, a single amino acid change in the ligand binding pocket formed by the 7 transmembrane segments of the rhodopsin receptor (Lys296Glu) leads to constitutive receptor activity and the pathology termed retinitis pigmentosa; Keen et al., 1991 Genomics 11:199- 205. Mutations in GPCRs and disease have been extensively reviewed by Coughlin, S.R., 1994 Current Biology 6:191-197, and Spiegel, A.M., 1995 Ann. Rev. Physiol. 58:143-170.

It would be desirable to identify other GABAB receptors which could be used in the identification of modulators of the GABAB receptor, particularly compounds that could perhaps combat some of the significant pharmacological activities that have been found to be associated with GABAB receptor activation such as various CNS disorders, drug addiction, neuropathic pain and gastroesophogeal reflux and incontinence.

SUMMARY OF THE INVENTION

This invention relates to a novel γ-aminobutyric acid type B (GABAB) receptor polypeptide designated Gblc. In particular the present invention relates to human Gblc, illustrated in SEQ ED NO:2 and, more specifically, to the sequence of amino acids 18-899 of SEQ ID NO:2. The particular GABAB receptor sequence taught herein comprises a glutamine at position 21 in place of a histidine.

Accordingly, those GABAB receptor polypeptides of the gblc subgroup comprising this amino acid substitution are expressly included within the present invention.

A further aspect of this invention are isolated polynucleotides which encode the Gblc receptor polypeptides of the present invention. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. The nucleic acids may be any type of nucleic acid, with preferred forms being DNA

(genomic and cDNA) although this invention specifically includes RNAs as well.

Nucleic acid constructs may also contain regions which control transcription and translation such as one or more promoter regions, termination regions, and if desired enhancer regions. The nucleic acids may be inserted into any known vector including plasmids, and used to transfect suitable host cells using techniques generally available to one of ordinary skill in the art.

Another aspect of this invention are vectors comprising the above polynucleotides, and host cells comprising the vectors. Still another aspect of this invention is a method of making Gblc comprising introducing a vector comprising polynucleotides encoding Gblc into a host cell under culturing conditions.

Yet another aspect of this invention are assays for γ-aminobutyric acid type B (GABAB) receptor polypeptide modulators, e.g., agonists and antagonists

(compounds which stimulate or which inhibit the function of GABAB receptors). One type of assay in particular comprises determining whether binding occurs between a candidate compound and a receptor polypeptide comprising the extracellular region of the Gblc polypeptide. This can be carried out by means of a label directly or indirectly associated with the candidate compound. Another type of assay constituting part of the instant invention comprises determining if a candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide.

Assays which make up further aspects of this invention include binding assays such as competition for 125T_GABAB receptor agonist binding, coupling assays including agonist-mediated inhibition of forskolin-stimulated adenylate cyclase in cells expressing the Gblc receptor polypeptide, measurement of agonist-stimulated calcium release in cells expressing Gblc receptors and promiscuous G proteins such as G15/G16/Gqi5/Gqo5 such as aequorin assays and calcium-based dye assays, stimulation of inward rectifying potassium channels (GIRK channels, measured by voltage changes) in cells expressing Gblc receptors, and measurement of pH changes upon agonist stimulation of cells expressing Gblc receptors as measured with a microphysiometer. In certain cellular environments, stimulation of Gblc receptors may also lead to inhibition of calcium channels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows the nucleic acid (SEQ ID NO:l) and deduced amino acid (SEQ ID NO:2) sequences of human Gblc. The polypeptide sequence comprises a signal peptide (M1-A17), a single Sushi Repeat (T26-R98)_; two putative glycosylation sites (N24_; N& ), and exhibits weak amino acid identity (26% identity over 26 amino acid overlap) to selectins. The putative transmembrane domains, and their corresponding amino acid numbers, are as follows: TMl:Ser526-Phe550; TM2:Ser563-Gly589; TM3:Leu605-Trp625; TM4:Trp646-Ile665; TM5:Thr706- Ala726; TM6:His740-Thr761; and TM7:Gln767-Phe791. FIGURE 2 shows the amino acid sequence of human gblc (Genbank

Accession No. AJ012187; SEQ ID NO:8) aligned over the Gblc receptor polypeptide sequence of the instant invention.

FIGURE 3 shows the complete cDNA sequence of HG20 (SEQ ID NO:3). FIGURE 4 shows the complete amino acid sequence of HG20 (SEQ ID NO:4).

FIGURE 5 A shows the major schematic topology/structural domains of gbla, gblb and gblc receptors, namely, the signal peptide, Sushi repeats, coiled- coil and PDZ domains. Figure 5B illustrates the amino acid alignment of the extracellular N-termini of human gbla, gblb, and Gblc isoforms. The proposed signal peptide cleavage site of gbl is marked with scissors, and arrows delimit the Sushi domains (SU). An arrow (Ψ) marks the start of the LIV-BP-like domain. Gaps (...) were introduced to maximize alignment. The novel Gblc isoform differs from gbla by an in-frame 62 amino acid deletion, and elimination of one protein-protein interacting Sushi Repeat (also known as short consensus repeat) leaving a single Sushi Repeat interacting module. The N-terminus common to the gbl isoforms exhibits weak amino acid identity to the bacterial periplasmic binding protein LIV-BP and is predicted to form two lobes that bind ligand according to a Venus flytrap model for receptor activation. The entire N-terminal domain of gbl, in the absence of the other portions of the receptor, is sufficient to specify agonist and antagonist binding.

FIGURE 6 shows a summary of BMAX (estimated maximal receptor density) and Kd for gblc and Gblc from 3H-CGP71872 saturation binding.

FIGURE 7 shows how GABA mediated a dose-dependent inhibition of forskolin-stimulated cAMP (GABA EC50 value of 21±8 nM). Basal cAMP levels were approximately 1 pmol cAMP/2 X 10^ cells and forskolin-induced cAMP levels typically greater than 20-fold. The functional and stably expressed Gblc-gb2 heterodimer subtype exhibited nanomolar potency for GABA similar to the reported affinity of GABA at neuronal GABAB receptors.

DETAILED DESCRIPTION OF THE INVENTION

As used throughout the specification and claims, the following definitions apply:

"Functional GABAB receptor" refers to the receptor formed following co-expression of Gblc and a subunit known variously as GABAβR2 (White et al.,

1998 Nature 396:679-682; and Jones et al., 1998 Nature 396:674-679); GBR2 (Kuner et al., 1999 Science 283:74-77); or gb2 (Ng et al., 1999 J. Biol. Chem. 274;7607- 7610), also known as HG20 (International Patent Application PCT/US99/02361, filed February 3, 1999). A functional GABAB receptor, further, displays at least one functional response when exposed to GABAB receptor agonists such as GABA or (-) baclofen. Examples of functional responses are: pigment aggregation in Xenopus melanophores, modulation of cAMP levels, coupling to inwardly rectifying potassium channels, mediation of late inhibitory postsynaptic potentials in neurons, increases in potassium conductance, and decreases in calcium conductance.

"HG20 or a functional equivalent thereof refers to HG20 (or gb2) and those polypeptides capable of interacting with a Gblc polypeptide to form a heterodimer active as a functional GABAB receptor, e.g., GABAβR2, and GBR2.

In accordance with the instant invention, Applicants have cloned a novel cDNA from adult human cerebellum mRNA which encodes an isoform of the γ-aminobutyric acid receptor "gblc" previously published by GlaxoWellcome in Genbank, Accession No. AJ012187. The novel receptor, designated "Gblc", similarly encodes a protein of 899 amino acids, but differs by a single amino acid substitution (a glutamine in place of a histidine at position 21). This difference is found in the extracellular N-terminus of the gblc receptor which is entirely responsible for ligand binding. As a result, the novel isoform disclosed herein exhibits higher affinity for antagonist and is more similar to native neuronal GABAB receptors which exhibit, e.g., approximately 1 nM Kd for CGP71872.

The human polypeptide sequence of the Gblc polypeptide of the instant invention is provided in Figure 1. As noted above, the particular GABAB receptor sequence taught herein comprises a glutamine at position 21 in place of a histidine. Accordingly, those GABAB receptor polypeptides of the gblc subgroup comprising this amino acid substitution are expressly included within the present invention. Polypeptides of the gblc subgroup are readily distinguished structurally from those of the la and lb subgroups. Gblc subgroup polypeptides differ from gbla subgroup polypeptides by an in-frame 62 amino acid deletion, and elimination of one protein-protein interacting Sushi Repeat. Gblb subgroup polypeptides lack both N- terminus Sushi Repeats.

Gblc proteins contain various functional domains, including one or more domains which anchor the receptor in the cell membrane, and at least one ligand binding domain. As with many receptor proteins, it is possible to modify many of the amino acids, particularly those which are not found in the ligand binding domain, and still retain at least a percentage of the biological activity of the original receptor. Thus this invention specifically includes modified functionally equivalent Gblcs which have truncated, or mutated N-terminal portions. This invention also specifically includes modified functionally equivalent Gblcs which contain modifications and/or deletions in other domains, which are not accompanied by a loss of functional activity. Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention.

Particular embodiments of the instant invention are those truncated forms of Gblc which comprise the extracellular portion of the receptor (amino acids 18-525; 1-17 encodes the putative signal domain), but lack the intracellular signaling portion of the receptor, and to nucleic acids encoding these truncated forms. Such truncated receptors are useful in various binding assays. Receptor chimeras (i.e., fusion proteins) which contain modifications and/or deletions not accompanied by a loss of functional activity are also included within the instant invention.

Isolated polynucleotides encoding the above polypeptides also constitute an integral part of the instant invention.

A further aspect of the instant invention are vectors comprising the above polynucleotides. This includes a variety of expression vectors useful in effecting the expression of recombinant HG20 or Gblc. Commercially available expression vectors which are suitable in this capacity include, but are not limited to, pMClneo (Stratagene), pSG5 (Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC 37593), pBPV- 1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), and the PT7TS oocyte expression vector (or similar expression vectors containing the globin 5' UTR and the globin 3' UTR). The choice of vector will depend upon the cell type used, the level of expression desired, and the like.

Cells comprising the above vectors constitute a further aspect of the instant invention. Particularly in the assays of the instant invention, the present invention employs cells co-expressing HG20 and Gblc, allowing for the formation of functional GABAB receptors and HG20/Gblc heterodimers. Such cells are generally produced by transfecting cells that do not normally express functional GABAB receptors with either a single expression vector or independent vectors encoding HG20 and Gblc. The cells are then cultured under conditions such that heterodimers of HG20 and Gblc are formed where the heterodimers constitute functional GABAB receptors. In this way, recombinant host cells expressing GABAB receptors are produced. This does not exclude the use of cells endogenously expressing a functional GABAB receptor, however.

Host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cells and cell lines which are suitable for recombinant expression of HG20 and Gblc and which are commercially available, include but are not limited to, L cells L-M(TK^~ ) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), HΕK293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NTH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), Xenopus melanophores, and Xenopus oocytes. In the case of Xenopus oocytes, co-expression of HG20 and Gblc is often effected by microinjecting RNA encoding HG20 and RNA encoding Gblc into the oocytes rather than by transfecting the oocytes with expression vectors encoding HG20 and Gblc. Microinjection of RNA into Xenopus oocytes in order to express proteins encoded by the RNA is well known in the art.

Another aspect of the instant invention are the assays afforded by disclosure of the novel GABAB receptor, Gblc. These allow for the identification of modulators of the GABAB receptor, e.g., agonists and antagonists (i.e., compounds which stimulate or which inhibit the function of gamma-aminobutyric acid type B (GABAβ) receptors) potentially useful in the treatment and/or prevention of the various afflictions attributed to GABAB receptor function or lack thereof, including various CNS disorders, drug addiction, neuropathic pain and gastroesophogeal reflux and incontinence. Other types of modulators are inverse agonists and allosteric modulators of agonists.

One type of assay involves determining whether binding occurs between a candidate compound and a receptor polypeptide comprising the extracellular region (amino acids 18-525 of SEQ ID NO:2) of the Gblc GABAB receptor polypeptide. This expressly includes binding to soluble proteins lacking transmembrane and intracellular regions, cells or membrane preparations bearing a receptor polypeptide comprising the extracellular region, fusion proteins comprising the extracellular Gblc region and fragments of the Gblc polypeptide of SEQ ED NO: 2. The use of membranes rather than cells is well known in the art. Moreover, the above assay as well as all the assays disclosed relating to binding of ligand can be run with Gblc receptor polypeptide monomers. Determination and measurement of binding can be done by means of a label directly or indirectly associated with the candidate compound.

This assay as well as the other assays disclosed in the instant invention can further be carried out in the presence of a competitor, for instance, a GABAB receptor agonist.

Accordingly, the present invention includes a method for the identification of agonists or antagonists of the GABAB receptor which comprises exposing cells expressing HG20 or a functional equivalent thereof and a receptor polypeptide comprising the extracellular region of the Gblc GABAB receptor polypeptide to a suspected agonist or antagonist in the presence and in the absence of a known GABAB receptor agonist. In the presence of a GABAB receptor agonist or antagonist, the amount of binding of the known agonist will be less than that seen upon contact of the receptor with the known agonist. In preferred embodiments, the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide are recombinant cells that have been transfected with a vector or vectors comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide.

Optionally, control experiments can be run utilizing control cells that are the same as the cells above except that the control cells do not express functional GABAβ receptors. The amount of binding of GABAB receptor agonist to the control cells should be significantly less than the amount of binding of agonist to cells expressing functional GABAB receptor. In this way, one can ensure that the binding of agonist in the above assays is actually due to binding of the agonist to GABAB receptors. Therefore, if the compound can block this binding, the compound is also likely to bind to GABAB receptors.

Another type of assay constituting part of the instant invention is for the identification of compounds which modulate the function of GABAB receptors which comprises determining if a compound upon contact with a receptor polypeptide comprising the extracellular region of the Gblc receptor results in a signal generated by activation or inhibition of the polypeptide. This can be done using detection systems appropriate to the cells or cell membranes bearing the polypeptide. A signal is an event indicating a functional response of the GABAB receptor. Note, as above, this assay expressly includes use of soluble proteins lacking transmembrane and intracellular regions, cells or membrane preparations bearing a receptor polypeptide comprising the extracellular region, fusion proteins comprising the extracellular Gblc region and fragments of the Gblc polypeptide of SEQ ED NO:2.

One skilled in the art is familiar with a variety of methods of determining and measuring the functional responses of G-protein coupled receptors such as the GABAB receptor; e.g., through the monitoring of changes in pigment distribution in melanophore cells, through the monitoring of changes in cAMP or calcium concentration or chemotaxis, through the monitoring of changes in membrane currents in Xenopus oocytes, through the monitoring of changes in calcium concentration measured using the aequorin assay, or through the monitoring of changes in inositol phosphate levels. Depending upon the cells in which heterodimers of HG20 and Gblc are expressed, and thus the G-proteins with which the functional GABAB receptor is coupled, certain of such methods may be appropriate for measuring the functional responses of such functional GABAB receptors. It is well with the competence of one skilled in the art to select the appropriate method of measuring the functional response for a given experimental system. Note that while the functionality of Gblc is attributed to heteromer formation, it remains possible that GABAβ receptor monomers or homodimers are functional when in certain cellular environments.

Preferred embodiments of the present invention are wherein HG20 or a functional equivalent thereof is expressed along with the receptor polypeptide such that heterodimers of the receptor polypeptide and HG20 are allowed to form.

This assay as well as the assays above can further be carried out in the presence of a competitor, for instance, a GABAB receptor agonist.

Accordingly, the present invention includes assays by which GABAB receptor antagonists or inverse agonists may be identified by their ability to antagonize a functional response mediated by the GABAB receptor in cells. One such method comprises contacting cells expressing HG20 or a functional equivalent thereof and a receptor polypeptide comprising the extracellular region of the Gblc receptor polypeptide with a suspected antagonist or inverse agonist and determining whether a functional response follows. A GABAB receptor antagonist will antagonize GABAB receptor function whereas an inverse agonist will result in a function opposite of a GABAB receptor agonist (e.g., a decrease versus an increase in a specific function). A preferred embodiment of the present invention is the above assay wherein the cells are contacted with the suspected antagonist or inverse agonist in the presence of a known GABAB receptor agonist, and wherein the response is measured and compared to that of the known agonist. In this situation, the functional response of the known agonist would be less in the presence of a GABAB receptor antagonist or inverse agonist. Especially preferred embodiments comprise the further step of contacting a separate group of cells expressing the receptor polypeptide (the control group) with the suspected antagonist or inverse agonist; a lack of response in the control indicating the presence of an antagonist and a response in contradiction with (or in a direction opposite to) that of the known agonist indicating the presence of an inverse agonist. In preferred embodiments, the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide comprising the extracellular region of the Gblc receptor polypeptide are recombinant cells that were transfected with a vector or vectors comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide. The present invention further includes assays by which GABAB receptor agonists or allosteric modulators of agonists may be identified by their ability to promote a functional response mediated by the GABAB receptor in cells. One such method comprises contacting cells expressing HG20 or a functional equivalent thereof and a receptor polypeptide comprising the extracellular region of a Gblc receptor polypeptide with a suspected agonist of Gblc or an allosteric modulator of such and determining whether a functional response follows. Contact with a GABAB receptor agonist should stimulate GABAB receptor function. By contrast, contact with an allosteric modulator of an agonist should enhance stimulation brought on by an agonist. A preferred embodiment of the present invention is the above assay wherein the cells are contacted with the suspected agonist or allosteric modulator in the presence of a known GABAB receptor agonist, and wherein the response is measured and compared to that of the known agonist, the functional response being greater in the presence of a GABAB receptor agonist or allosteric modulator.

Especially preferred embodiments comprise the further step of contacting a separate group of cells expressing the receptor polypeptide (the control group) with the suspected agonist or allosteric modulator; a lack of response in the control indicating the presence of an allosteric modulator and a functional response indicating the presence of an agonist. In preferred embodiments, the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide are recombinant cells that were transfected with a vector or vectors comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide.

In particular embodiments, the functional response is selected from the group consisting of: modulation of the activity of an ion channel; changes in calcium concentration; changes in a signal from a reporter gene whose expression is controlled by a promoter that is induced by interaction of an agonist with the GABAB receptor; and changes in membrane currents. In particular embodiments, the change in membrane current is measured in Xenopus oocytes. In other embodiments, the change in membrane current is the modulation of an inwardly rectifying potassium current.

Particular types of functional assays that can be used to identify agonists or antagonists of GABAB receptors include transcription-based assays.

Transcription-based assays involve the use of a reporter gene whose transcription is driven by an inducible promoter whose activity is regulated by a particular intracellular event such as, e.g., changes in intracellular calcium levels that are caused by the interaction of a receptor with a ligand. Transcription-based assays are reviewed in Rutter et al, 1998, Chemistry & Biology 5:R285-R290. The transcription-based assays of the present invention rely on the expression of reporter genes whose transcription is activated or repressed as a result of intracellular events that are caused by the interaction of an agonist such as baclofen with a heterodimer of HG20 and Gblc where the heterodimer forms a functional GABAB receptor. In the above assays, compounds capable of effecting a greater signal from the reporter gene than GABAβ receptor agonist are noted as more potent agonists than that employed and warrant further study. Conversely, if a suspected antagonist is utilized along with agonist, the inquiry would be whether the cells treated with the agonist emitted a greater signal than the cells treated with the agonist and the suspected antagonist, in which case the compound would be an antagonist of the GABAB receptor. Suitable reporter genes for use in the above assays are green fluorescent protein (GFP), β- galactosidase, and luciferase.

A sensitive transcription based assay in accordance with the instant invention employs a plasmid encoding β-lactamase under the control of an inducible promoter. This plasmid is transfected into cells together with a plasmid encoding a receptor for which it is desired to identify modulators such as agonists or allosteric modulators of agonists. The inducible promoter on the β-lactamase is chosen so that it responds to at least one intracellular signal that is generated when an agonist or modulator of same binds to the receptor. Thus, following such binding of agonist or modulator to receptor, the level of β-lactamase in the transfected cells increases. This increase in β-lactamase is measured by treating the cells with a cell-permeable dye that is a substrate for cleavage by β-lactamase. The dye contains two fluorescent moieties. In the intact dye, the two fluorescent moieties are close enough to one another that fluorescence resonance energy transfer (FRET) can take place between them. Following cleavage of the dye into two parts by β-lactamase, the two fluorescent moieties are located on different parts, and thus can drift apart. This increases the distance between the fluorescent moieties, thus decreasing the amount of FRET that can occur between them. It is this decrease in FRET that is measured in the assay. Through the use of the above assay wherein the inducible promoter that drives β-lactamase is activated by an intracellular signal generated by the interaction of the GAB Aβ receptor and modulators thereof, one of ordinary skill in the art could identify modulators of GABAB receptors such as agonists, allosteric modulators of agonists, inverse agonists, and antagonists. To produce the GABAβ receptor, a plasmid encoding HG20 and a plasmid encoding Gblc are transfected into the cells. Cells endogenously expressing functional GABAB receptor are also suitable in the instant assay. The cells are exposed to the cell-permeable dye and then exposed to compounds suspected of being agonists of the GABAB receptor. Those compounds that cause a decrease in FRET are likely to actually be agonists of the GABAβ receptor. To determine if the compounds that are identified as agonists are sufficiently potent agonists to warrant further study, one could compare the decrease in FRET caused by the compounds to the decrease in FRET that is caused by baclofen when baclofen is substituted for the compound in the assay. Accordingly, the present invention includes a method for identifying modulators of the GABAB receptor comprising: (a) providing cells expressing (i)

HG20 or a functional equivalent thereof, (ii) a receptor polypeptide comprising the extracellular region of Gblc, and (iii) β-lactamase under the control of an inducible promoter that is activated by an intracellular signal generated by the interaction of GABAβ receptor agonist and the GABAB receptor; (b) exposing the cells to a substrate of β-lactamase that is a cell-permeable dye that contains two fluorescent moieties where the two fluorescent moieties are on different parts of the dye and cleavage of the dye by β-lactamase allows the two fluorescent moieties to drift apart; and (c) measuring the amount of fluorescence resonance energy transfer (FRET) in the cells following contact with a suspected GABAB receptor modulator. In the instance wherein agonists are sought, the amount of FRET can be compared with that measured in the absence of the suspected agonist to obtain a value for the decrease in FRET caused by the suspected agonist. A decrease in FRET resulting from a known GABAB receptor agonist can be determined by measuring the amount of FRET in the presence and in the absence of the agonist. By comparing the decrease in FRET caused by the suspected agonist to the decrease in FRET caused by the known agonist, one can identify modulators, and agonists in particular, of the GABAB receptor. An agonist would exhibit an equal or greater decrease in FRET than the known agonist.

In fact, by comparing the decrease in FRET caused by a suspected agonist to the decrease in FRET caused by a known GABAβ receptor agonist such as baclofen, one can estimate how potent an agonist the suspect compound is. If the decrease in FRET caused by the compound is larger than the decrease in FRET caused by the agonist, then the compound is likely to be a more potent agonist than the known agonist employed. If the decrease in FRET caused by the compound is about the same as the decrease in FRET caused by the known agonist, then the compound is likely to be an agonist of about the same potency as that employed. If the decrease in FRET caused by the compound is less than the decrease in FRET caused by the known agonist, then the compound is likely to be a weaker agonist than that employed.

The above-described assay can be further modified to an assay for identifying antagonists of the GABAB receptor. This method comprises (a) providing cells expressing (i) HG20 or a functional equivalent thereof, (ii) a receptor polypeptide comprising the extracellular region of Gblc, and (iii) β-lactamase under the control of an inducible promoter that is activated by an intracellular signal generated by the interaction of GABAβ receptor agonist and the GABAB receptor;

(b) exposing the cells to a substrate of β-lactamase that is a cell-permeable dye that contains two fluorescent moieties where the two fluorescent moieties are on different parts of the dye and cleavage of the dye by β-lactamase allows the two fluorescent moieties to drift apart; and (c) measuring the amount of fluorescence resonance energy transfer (FRET) in the cells following contact with a suspected GABAB receptor antagonist. The amount of FRET can further be measured upon contact with a known GABAβ receptor agonist and the suspected antagonist. This value can then be compared with the value obtained from contact with a known GABAB receptor agonist; a lesser amount of FRET in the cells contacted with agonist indicating the presence of an antagonist.

In a particular embodiment of the above-described methods, the inducible promoter that is activated by at least one intracellular signal generated by interaction of an agonist with the GABAB receptor is a promoter that is activated by changes in membrane currents, e.g., changes in potassium currents or activation of the map kinase signaling pathway. In other embodiments, the inducible promoter is activated by the transcription factor NFAT, or is activated by a signal transduced by a chimeric Gq protein, or a signal generated by protein kinase C activity, or by changes in intracellular calcium levels.

The assays described above can be further modified to an additional assay for identifying antagonists of the GABAB receptor. Such modification would involve the use of β-lactamase under the control of a promoter that is repressed by at least one intracellular signal generated by interaction of a GABAB receptor agonist with the GABAB receptor and would also involve running the assay in the presence of said agonist. When the cells are exposed to compounds suspected of being antagonists of the GABAB receptor, β-lactamase will be induced, and FRET will decrease, if the compound tested is able to counteract the effect of the agonist, i.e., if the compound tested is actually an antagonist.

In a particular embodiment, the inducible promoter that is repressed by at least one intracellular signal generated by interaction of a GABAB receptor agonist with the GABAβ receptor is a promoter that is repressed by changes in potassium currents. In particular embodiments of the above-described methods, β- lactamase is TEM-1 β-lactamase from Escherichia coli.

In other embodiments, the substrate of β-lactamase is CCF2/AM (Zlokarnik et al., 1998, Science 279:84-88). In particular embodiments of the above-described methods, the cells express a promiscuous G-protein, e.g., Gαl5 or G l6. In other embodiments, the cells have been transfected with an expression vector that directs the expression of a G-protein subunit or subunits.

The assays described above could be modified to identify inverse agonists. In such assays, one would expect a decrease in β-lactamase activity where agonists would produce an increase.

The assays described above could also be modified to identify allosteric modulators of GABAB receptor agonists. In such assays, one would not expect a response from the modulator in the absence of an agonist. In particular embodiments of the above-described methods, HG20 is a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:4; positions 9-941 of SEQ ID NO:4; positions 35-941 of SEQ ID NO:4; positions 36-941 of SEQ ID NO:4; positions 38-941 of SEQ ID NO:4; positions 39-941 of SEQ ED NO:4; positions 42-941 of SEQ ID NO:4; positions 44- 941 of SEQ ID NO:4; positions 46-941 of SEQ ID NO:4; positions 52-941 of SEQ ID NO:4; positions 57-941 of SEQ ED NO:4; the amino acid sequence encoded by the DNA sequence deposited in GenBank Accession No. AF056085; the amino acid sequence encoded by the DNA sequence deposited in GenBank Accession No. AJ012188; and the amino acid sequence encoded by the DNA sequence deposited in GenBank Accession No. ASF074482.

It is important to note that in all of the above assays, HG20 (or gb2)can be substituted with a functional equivalent such as GABAβR2, or GBR2.

In particular embodiments of the above-described methods, HG20 is a chimeric HG20 protein. By chimeric HG20 protein is meant a contiguous polypeptide sequence of HG20 fused in frame to a polypeptide sequence of a non- HG20 protein. For example, the N-terminal domain and seven transmembrane spanning domains of HG20 fused at the C-terminus in frame to a G protein is a chimeric HG20 protein. Another example of a chimeric HG20 protein is a polypeptide comprising the FLAG epitope fused in frame at the amino terminus of amino acids 52-941 of SEQ ED NO:4. Especially preferred forms of chimeric HG20 proteins are those in which a non-HG20 polypeptide replaces a portion of the N- terminus of HG20. Chimeric Gblc proteins or fragments of the GABAβ receptor polypeptide Gblc may also be used in the present invention. In particular embodiments, the chimeric Gblc protein comprises the extracellular region of Gblc with or without the signal sequence. In particularly preferred embodiments, a polypeptide comprising the extracellular region of Gblc (amino acids 18-525 of SEQ ED NO:2) is fused to another non-Gblc protein or portion thereof.

In preferred embodiments, the expression vector encoding HG20 comprises a nucleotide sequence selected from the group consisting of: positions 293- 3,115 of SEQ ID NO:3; positions 317-3,115 of SEQ ID NO:3; positions 395-3,115 of SEQ ID NO:3; positions 398-3,115 of SEQ ID NO:3; positions 404-3,115 of SEQ ID NO:3; positions 407-3,115 of SEQ ID NO:3; positions 416-3,115 of SEQ ID NO:3; positions 422-3,115 of SEQ ID NO:3; positions 428-3,115 of SEQ ID NO:3; positions 446-3,115 of SEQ ID NO:3; and positions 461-3,115 of SEQ ID NO:3. Some of the above-described methods can be modified to take advantage of other ways of assaying for agonist activity at the GABAB receptor.

Agonists/antagonists/inverse agonists may affect the internalization or trafficking of functional GABAB receptors. For example, in the case of the β2-adrenergic receptor, agonist exposure results in receptor internalization. Therefore, receptor trafficking between intracellular pools and the cytoplasmic membrane may be considered an assay of agonist activity. It may be that GABAB receptor trafficking is modulated by agonists in a similar manner. It would then be possible to identify agonist activity by monitoring GABAB receptor trafficking. Such trafficking can be monitored by whole cell immunohistochemistry and confocal microscopy or by surface and intracellular receptor labeling and flow cytometry.

Furthermore, because the functional GABAB receptor may be a heterodimer, then agonists/antagonists/inverse agonists may be expected to alter the ratio of heterodimer to monomer. Hence the disruption or appearance of a heterodimer may be considered an additional screening assay. In this case, the monitoring of receptor dimerization or disappearance may be done by the appearance or disruption of FRET. Each of the monomers are labeled with a fluorophore such that close proximity would allow FRET to occur. Upon agonist binding, one might see disruption of FRET, indicating disruption of dimers or increase in FRET indicating more dimerization in the course of agonist activation.

While the above-described methods are explicitly directed to testing whether "a" compound is an agonist or antagonist of the GABAB receptor, it will be clear to one skilled in the art that such methods can be adapted to test collections of compounds, e.g., combinatorial libraries, collections of natural produces, etc., to determine whether any members of such collections are activators or inhibitors of the GABAβ receptor. Accordingly, the use of collections of compounds, or individual members of such collections, as the compound in the above-described methods is within the scope of the present invention.

Agonists and antagonists identified by the above-described methods are useful in the same manner as well-known agonists and antagonists of GABAB receptors. For example, (-)baclofen is a known agonist of GABAB receptors and, in racemic form, is a clinically useful muscle relaxant. Gabapentin has been sold since 1994 in the United States as a treatment for epilepsy. The therapeutic potential of GABAβ receptor agonists and antagonists is well documented. For agonists, the therapeutic potential is said to include use as muscle relaxants and anti-asthmatics. For antagonists, the therapeutic potential is said to include use as antidepressants, anticonvulsants, nootropics, and anxiolytics. Given the wide range of utility displayed by known agonists and antagonists of GABAβ receptors, it is clear that those skilled in the art would consider the agonists and antagonists identified by the methods of the present invention to be pharmacologically useful. In addition, it is believed that such agonists and antagonists will also be useful in the treatment of epilepsy, neuropsychiatric disorders, and dementias.

The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLE 1 Construction of GABAB Receptor Expression Constructs

The human gblc isoform was obtained from human cerebellum cDNA (Clontech) by nested PCR cloning using a High-fidelity HF-PCR cloning kit (Clontech). The first step of amplification used primers: hgblcF-ECORl 5'-GAT ATC GAA TTC GCC ACC ATG TTG CTG CTG CTG CTA CTG GCG CCA CTC- 3' (SEQ ID NO:5) and gblRR 5'-gc cct tec cct etc cct ttc cct ccc-3' (SEQ ID NO:6). PCR conditions were: precycle denaturation of 94°C for 15s, followed by denaturation at 94°C for 15s and an annealing/extension step at 68°C for 4 min for 30 cycles. A final extension step was done at 72°C for 3 min. The second amplification used primers: hgblcF-ECORl and hgblECORl-R 5'-GCC GAG GAA TTC TCA CTT ATA AAG CAA ATG CAC TCG ACT CCC (SEQ ID NO:7). PCR conditions were as described above. PCR products were subcloned into PCR-TA cloning vector (Invitrogen), pT7 vector for expression in Xenopus oocytes, pcDNA3.1/Zeo(+) (Invitrogen) for transient high-level expression in COS-7 cells, or pERES-bleomycin bicistronic vector (Clontech) for stable expression in HEK-293 cells.

EXAMPLE 2

Transient Expression for Radioligand binding

COS-7 (ATCC) cells were grown to -70% confluence. 6μg of Glaxo lc or Merck lc plasmid DNA (Qiagen) were transfected into 1.2 million COS-7 cells using 36μl of lipofectamine as recommended by manufacturer (Gibco BRL).

EXAMPLE 3 Receptor Binding

Cells were harvested 72hr-post transfection. Crude membrane preparations were prepared as described in Belley et al., 1999 Bioorganic & Medicinal Chemistry 7:2697-2704. ³H-CGP71872 (a GABAB receptor antagonist) was custom synthesized by NEN. Saturation binding experiments were conducted using 25μg of membrane and increasing concentration of radiolabeled 3H-CGP71872 ranging from ~0.05nM to 75nM. Non-specific binding was obtained in the presence of lμM cold CGP71872. Each condition was made in duplicate. Incubation was carried out in 200μl of binding buffer (50mM Tris, pH7.4, 2.5mM CaCl₂, IX protease inhibitor cocktail - Complete Tablet™ (Boehringer Mannheim)) with constant shaking at room temperature for 2hr. Brandel harvester was used to trap bound ligand onto Whatman GF/B filters. Data analysis was performed using GraphPad Prism (San Diego).

EXAMPLE 4 Transient Expression and Generation of Gbl-Gb2 Stable HEK-293 Cell Lines

Receptor DNAs (6 μg total DNA/1.2 X 10⁶ cells) were transfected into COS-7 or HEK-293 cells using 36 μl Lipofectamine reagent (Gibco BRL) according to the manufacturer's instructions. Stable gb2-expressing HEK-293 clones were selected by growth in puromycin (5μg/ml) containing media and dilution cloning. RNA was prepared from 95 clones using Trizol reagent (GibcoBRL) and 10 μg total RNA spotted by vacuum using a dot-blot apparatus onto BrightStar-Plus nylon membranes (Ambion). The blot was hybridized with a ³²P-labeled DNA fragment encoding the full-length gb2 receptor (10° cpm/ml) in Zip-Hyb solution (Ambion) for 10 h at 50 °C, and washed at 55 °C for 90 min in high-stringency wash buffer. Two high gb2 receptor RNA-expressing clones (gb2.46, and gb2.10) were analysed for cell surface gb2 receptor expression by indirect staining using gb2 antisera 1630.1-1630.2 and goat anti-rabbit antibodies coupled with Alexa-488 on a Becton Dickinson FACS Vantage SE flow-cytometer configured to detect FITC fluorescence. Gbl isoform DNAs were transfected into the gb2.10 clone as described, and bulk gblc- gb2 stables were selected by growth in puromycin (5μg/ml) and phleomycin (50 μg/ml) containing media. Control stable pIRES -puromycin and pE ES-bleomycin vector expressing HEK-293 cell lines were generated also by growth in antibiotic selection media.

EXAMPLE 5 cAMP Functional Assays

cAMP determinations were made using a scintillation proximity assay (SPA) kit (Amersham, ONT). Briefly, HEK-293 cells were washed, detached, and 77,000-100,000 cells/well resuspended in Hank's Balanced Salt Solution containing 25 mM HEPES pH 7.4, 100 μM 4-(3-butoxy-4-methoxybenzyl)-2-imadazolidinone (Ro 20-1724, BIOMOL, PA) and incubated for 20 min at 37 °C. 2 μM forskolin and ligands (10^"9-10 ³ M) were added and incubated for 30 min at 37 °C. Cells were lysed by boiling and cAMP levels were determined by SPA according to the manufacturer's instructions. Data were analyzed by nonlinear least-squares regression using the computer-fitting program GraphPad Prism version 2.01 (San Diego).

EXAMPLE 6 In Vitro Receptor Expression

In vitro coupled transcription/translation reactions were performed in the presence of [ SJmethionine in the TNT® Coupled Reticulocyte Lysate system (Promega, Madison, WI) using pcDNA3.1 plasmids containing the gbla, gblb, and gblc DNAs. Translation products were analysed by electrophoresis on 8-16% Tris- Glycine SDS gradient gels (Novex pre-cast gel system, San Diego, CA) under denaturing and reducing conditions. Gels were fixed, dried and exposed to Kodak X- AR film at -70 °C for 4 to 24 h.

Claims

WHAT IS CLAIMED:

1. An isolated human gamma-aminobutyric acid type B (GABAβ) receptor polypeptide comprising the amino acid sequence of SEQ ED NO:2.

2. An isolated human gamma-aminobutyric acid type B (GABAβ) receptor polypeptide comprising amino acids 18-899 of SEQ ED NO:2.

3. An isolated human gamma-aminobutyric acid type B

(GABAβ) gblc receptor polypeptide comprising a glutamine in place of a histidine at a position corresponding to position 21 of the GABAB receptor polypeptide of claim 1.

4. A receptor polypeptide comprising the extracellular portion of human gamma-aminobutyric acid type B (GABAB) receptor polypeptide of SEQ ID

NO:2.

5. A receptor polypeptide in accordance with claim 4 wherein the extracellular portion is fused with another protein or fragment thereof.

6. A receptor polypeptide in accordance with claim 4 wherein the receptor is a soluble GABAB receptor.

7. A receptor polypeptide in accordance with claim 4 comprising amino acids 1-525 of SEQ ID NO:2.

8. A receptor polypeptide in accordance with claim 4 comprising amino acids 18-525 of SEQ ED NO:2.

9. An isolated polynucleotide encoding a GABAβ receptor polypeptide in accordance with claim 1.

10. A polynucleotide in accordance with claim 9 which is SEQ ID NO:l.

11. An isolated polynucleotide encoding a GABAB receptor polypeptide in accordance with claim 2.

12. A polynucleotide in accordance with claim 11 comprising nucleotides 52-2697 of SEQ ID NO:l.

13. An isolated polynucleotide encoding a receptor polypeptide in accordance with claim 4.

14. A polynucleotide in accordance with claim 13 comprising nucleotides 1-1575 of SEQ ED NO:l.

15. A polynucleotide in accordance with claim 13 comprising nucleotides 52-1575 of SEQ ID NO: l.

16. A vector comprising the polynucleotide of claim 10.

17. A vector comprising the polynucleotide of claim 12.

18. A vector comprising the polynucleotide of claim 14.

19. A vector comprising the polynucleotide of claim 15.

20. A host cell comprising the vector of claim 16.

21. A host cell comprising the vector of claim 17.

22. A host cell comprising the vector of claim 18.

23. A host cell comprising the vector of claim 19.

24. A method for the identification of modulators of gamma- aminobutyric acid type B (GABAB) receptors which comprises:

(a) contacting a receptor polypeptide in accordance with claim 8 with a candidate compound, and (b) determining if binding occurs; binding indicating the presence of a GABAB receptor modulator.

25. The method of claim 24 wherein the binding is measured in the presence of a competitor.

26. The method of claim 24 wherein the modulators are agonists or antagonists.

27. The method of claim 24 which comprises exposing cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide to a suspected agonist or antagonist in the presence of a known GABAB receptor agonist; the amount of binding of the known agonist being less in the presence of an agonist or antagonist of the GABAβ receptor.

28. The method of claim 25 wherein the GABAB receptor agonist is baclofen.

29. The method of claim 27 wherein the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide are recombinant cells that were transfected with a vector or vectors comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide.

30. A method for the identification of compounds which modulate the function of gamma-aminobutyric acid type B (GABAB) receptors which comprises determining whether a candidate compound upon contact with a receptor polypeptide of claim 8 results in a signal generated by activation or inhibition of the polypeptide.

31. The method of claim 30 wherein HG20 or a functional equivalent thereof is expressed along with the receptor polypeptide such that heterodimers of the receptor polypeptide and HG20 are allowed to form.

32. The method of claim 30 wherein the method is done in the presence of a competitor.

33. The method of claim 32 wherein the competitor is baclofen.

34. A method in accordance with claim 30 for the identification of

GABAB receptor antagonists or inverse agonists which comprises:

(a) contacting cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide with a suspected antagonist or inverse agonist of the receptor polypeptide, and (b) determining whether a functional response follows.

35. The method of claim 34 wherein the cells are contacted with the antagonist or inverse agonist in the presence of a known GABAB receptor agonist, and wherein the response is measured and compared to that of a control, the control comprising the known agonist; the functional response of the known agonist being less in the presence of a GABAB receptor antagonist or inverse agonist.

36. The method of claim 35 wherein the response is compared to that of a control, the control comprising contacting a separate group of cells with the antagonist or inverse agonist; a lack of response in the control indicating the presence of an antagonist and a response in contradiction with that of the known agonist indicating the presence of an inverse agonist.

37. The method of claim 34 wherein the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide are recombinant cells that were transfected with a vector or vectors comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide.

38. A method in accordance with claim 30 for the identification of GABAβ receptor agonists or allosteric modulators of agonists which comprises:

(a) contacting cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide with a suspected agonist of the receptor polypeptide or an allosteric modulator of such, and

(b) determining whether a functional response follows.

39. The method of claim 38 wherein the cells are contacted with the agonist or allosteric modulator in the presence of a known GABAB receptor agonist, and wherein the response is measured and compared to that of a control, the control comprising contact of the receptor polypeptide with the known agonist; the functional response being greater in the presence of a GABAB receptor agonist or allosteric modulator.

40. The method of claim 39 wherein the response is compared to that of a control, the control comprising contacting a separate group of cells with the agonist or allosteric modulator; a lack of response in the control indicating the presence of an allosteric modulator and a functional response indicating the presence of an agonist.

41. The method of claim 38 wherein the cells expressing HG20 or a functional equivalent thereof and the receptor polypeptide are recombinant cells that were transfected with a vector comprising a polynucleotide(s) encoding one or both of the HG20 or the receptor polypeptide.