CA2196997A1 - Glutamate receptor - Google Patents

Abstract

The present invention relates to a human metabotropic glutamate receptor (hmGluR) subtype, isolated nucleic acids coding therefor, host cells producing a protein of the invention, methods for the preparation of such protein, nucleic acids and host cells, and uses thereof. Furthermore, the invention provides antibodies directed against a hmGluR protein of the invention.

Description

WO 96/06167 ~ rl ~ ,o ~ I~ r. ~. ,_, ~

Glutamate Receptor The present invention relates to a human mP~qhotropic glutamate receptor (hmGluR) subtype, isolated nucleic acids coding therefor, host cells producing a protein of the invention, methods for the preparation of such protein, nucleic acids and host cells, and uses thereof. r the invention provides antibodies directed against a hmGluR
protein of the invention.

M ~ Ibvl vl. r glutamate receptors (hmGluR) belong to the class of G-protein (guanine nucleotide binding protein) coupled receptors which upon binding of a O ~i~ligand may transduce an . " ' signal via an ~ ~ r second messenger system such as calcium ions, a cyclic nucleotide, d;C~JIOI~ UL inositol 1,4,5-i, ' , ' ~ into a ivlûoi~.al response. Possessing seven putative i ' spanning segments, preceded by a LYrge . " ' ; ~ ' dûmam and followed by a large carbo~cy~terrninal domain ' -r- glutamate receptors are ~ I' -- ,-- 1 . ;,. ~ by a common structure. Based on the degree of sequence identity at tbe amino acid level the class of mGluR can be divided into different ! ' ~ . ~ ~ g individual receptor subtypes ~akanishi, Science 258, 597-603 (1992)). Each mGluR subtype is encoded by a unique gene. Regarding the homology of an individual mGluR subtype to another subtype of a different subfamily, the arr~ino acid sequences are less than about 50 % identical.
Wlthin a subfamily the degree of sequence identity is generaUy less than about 70 9~O. Thus a parlicular subtype may be c~ . i ,. d by its amino acid sequence homology to another mGluR subtype, especially a subtype of the same _l- species. r. . a particular subtype may be ~ ; d by its region and tissue ~ its cellular and --'--" ' expression pattem or by its distinct IAl~.,;oloO;cal profile, e.g. by its cleCIlu~ iulog;~ and pl~- ,- "log;. ~l properties.
. .
The amino acid ~glutamate being the major excitatory ~..,. g' g;c systems are presumed to play an important role in numerous neuronal processes including fast excitatory synaptic i regulation of , releases, long-temm . Ieaming and memory, .1~ , 1 synaptic pLasticity, hypoxic-ischemic damage and neuronal cell death, erilpr~if~rm seizures, as well as the ~ r.O~ of several u~ ., disorders. Up to today, no; -f ~ ~IiO. iS available on human~ bu~lu~i~, glutamate receptor (hmGluR) subtype 2, e.g. on the amino acid sequence or tissue .' ~ This lack of knowledge p~Li.,uLul~ hampers the search for human Lh.,~ agents capable of specificaUy '' v any disorder Ym;l, l~l,lr to a defect .

WO 96/06167 1~ ~,l/~r . /~D
21 969q7 -2-=
', ~ i I I ~ ! ' in the v- Sic system. In view of the potential ~/LJD;ologiCdl and p ~h~lf~gif Al ~i g ~ of ~ ,~buLIup;c glutamate receptors, there is a need for human receptor subtypes and cells producing such subtypes in amounts sufficient for elucidating the clf ~ u~hJDiulogical and ,u~ properties of these proteins. For example, drug screening ~says require a purified human receptor protein in an active form. which has not yet been attainable.

It is an object of the present invendon to fulfll this need, namely to provide hmGluR
subtype 2, a nucleic acid coding therefor and host celis producing such subtype.HmGluR2 is potently activated by (2S,3S,4S)-oc-(.,,~l,uAy~,y~,lu~lu,u~l)-glycine ~L,CCG-I) and, when e~pressed e.g. in Chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells, negatively coupled to adenylate cyclase via G protein. Using a system -' . ' E, a I hmGluR subtype of the invention in screening for hmGluR
reactive drugs offers (among others) the ~ ' " of attaining a greater number of receptors per cell giving greater yield of reagent and a higher signal to noise ratio in assays as well as increased receptor subtype specificity (~Iu~.ldill~ resuldng in greater biological and disea~ specificity).

More b~,iG~,~lly, the pre~nt invendon relates to hmGluR2 having the amino acid ~quence depicted in SEQ ID NO:2.

According to the invendon the expression "hmGluR subtype" refers to a purified protein which belongs to the class of G protein-coupled receptors and which upon binding of a ,, ,, ligand transduces an f"r~rAf'f'11 ~ signal via an ~- ' second mes~nger system. In such case, the subtype of the invendon is .1. A. ~ ; ; in that it modifies the level of a cyclic nucleodde (cAMP, cGMP). Alt~dli~"ly, signal ~ may occur via direct interaction of the G protein coupled to the receptor subtype of the invendon with another membrane protein, such as an ion channels.
HmGluR2 is believed to be encoded by a disdnct gene which does not encode another " " 1 l ,u~, u~ glutamate receptor subtype. A patticular subtype may be ~ L ~ ;~- d by its disdnct l)llJDIUIUg;.~fl profile, preferably by its signal ' and ~ ol(,g;- ~1 properties. Pl --, - ..lf~;; I properdes are e.g. the ~lecdvity for agonists and antagonist responses.

As defined herein, a ,, ~;;c ligand is e.g. L,glutamate or another compoundinreracting with, and p~ Ih,ukuly binding to, a hmGluR subtype in a glutamate like ~ WO 96/06167 ~ ! 9 ~ 9 9 7 . i manner, such as ACPD (lS,3R-1 . J.E~ -1,3-dil~ u~ylic acid), an ~ ACPD-like ligand, e.g. QUIS (q . ' ), L-2-amino-4~ acid (AP4), L-CCG-L and the like. Other ligands, e.g. (RS)-a ,1 ~ cO,b~ lglycine (MCPG) or a-methyl-L-AP4, may interact wieh the receptor of the invention in such a way that binding of a ~' ~- Iigand is prevented.

As used I . . ~ .. r. or I - r , the terms "purified" or "isolated" are intended to refer to a molecule of the invention in an enriched or pure form obtainable from a natural source or by means of genetic; ~ g The purified protein, DNA and RNA of the invention may be useful in ways that the protein, DNA and RNA ~ they naturaUy occur are not, such as ;-1 ~ A of IL ' selectively - ~ ' g the expression or the activity of the hmGluR of the invention.

Purif1ed hmGluR of the invention means hmGluR2 which has been identified and is free of one or more . , of its natural c..~ Purified hmGluR includes purified hmGluR of the invention in ' ~ ceU culture. The enriched form of the subtype refers to a l,. ", ,~m~l~ containing said subtype in a ~ higher than natural, e.g. a ceUular membrane fraction comprising said subtype. E the subtype is in a pure form it is "~, free from other ~ l ' ' . L ' '~ from naturally occurring L - If desired, the subtype may be s~hlhili7~tl A preferred purified hmGluR2 of the invention is a ' protein. Preferably, the subtype of theinvention is in an active state mear~ing that it has boeh ligand binding and signal activity. Receptor activity is measured according to methods known in the art, e.g. using a binding assay or a functional assay, e.g. an assay as described below.

The invention is further intended to include variants of the receptor subtype of the invention. For example, a variant of the hmGluR subtype of the invention is a functional or ~ ' O ' equivalent of said sub~pe. A functional equivalent is a human protein displaying a yhJD;ulOo;~ profile essentiaUy identical to the profile ~ r ;~1 ~. of the hmGluR2 having the amino acid sequence set forth in SEQ ID NO:2. r. . u h . ., ., ~ I . a functional equivalent has more than 70 %, preferably more than 90 %, sequence identity with the protein having the amino acid sequence set forth in SEQ ID NO 2. Accordingly, a functional equivalent does not include another hmGluR subtype of the same subfamily, e.g. hmGluR3. The pl..~;olog;c.ll profile in vitro and in vivo includes receptor effector function, elc~ uyhJ.~iùloOi~ ~l and L ' ~,' ' properties, e.g. selective interaction with agonists or - ~ Exemplary funcdonal equivalents may be splice variants _ _ _ , _ _ _ _ _ _ _ _ _ _ wos6/06167 2, 96qq7 4 encoded by mRNA generated by alternative splicing of a primary transcript, amino acid mutants and gly~,v~LILiu.. variants. An ' ~g ' equivalent of the hmGluR2 having the amino acid sequence set forth in SEQ ID NO:2 is a protein or peptide capable of generating antibodies specific for said subtype. PorLions of the P~ domain of the receptor, e.g. peptides consisting of at least 6 to 8 amino acids, p~uLi~,ukuly about 20 amino acids, are considered I ' 1~ useful ~ ' ~g ' , h Further variants included herein are ' bound and soluble fragments and covalent or agO.~ .Li~ conjugates with other chemical moieties, these variants displaying one or more receptor functions, such as ligand binding or signal i ' The fragments of the invention are obtainable from a natural source, by chemical synthesis or by -- - - --' techniques. Due to their capability of competing with the ~ i O
L of the hmGluR subtype of the invention for its ~ ~~e, --- ~ , ligand, fragments, or derivatives thereof, comprising the ligand binding domain are envisaged as therapeudc agents.

Covalent derivatdves include for example aliphadc esters or amides of a receptor carbo~cyl group, O-acyl derivatives of hydro~yl group containing residues and N-acyl derivadves of amino group containing residues. Such derivadves can be prepared by linlcage of '- 1- - to reactable groups which are found in the side chains and at the N- andC-terminus of the receptor protein. The protein of the invention can also be labeled with a detectable group, for example " -' -~ ' 1, covalently bound to rare earth chelates or conjugated to a fluorescent moiety.

Further derivadves are covalent conjugates of a protein of the invendon with another protein or peptide (fusion proteins). E~amples are fusion proteins, . g different portions of different glutamate receptors. Such fusion proteins may be useful for changing the coupling to G-proteins and/or improving the sensidvity of a functional assay. For example, in such fusion proteins or cbimeric receptors, the ~ ' ' domains of thesubtype of the invendon may be replaced with the c ~ E. domains of anothermGluR subtype, p~uLi-,ulally a hmGluR subtype, e.g. a hmGluR subtype belonging to another subfamily. F ' 1~, suitable for the: u~,Lio.. of such a cbimeric receptor are the ~ ~I ' domains of a receptor which activates the ~,h~ , C/Ca2+
signahing pathway, e.g. mGluR1 (~5asu et al., Nature 349, 760-765) or mGluR5. Anin~rPll ' domain suitable for such an exchange is e.g. the second inrr~Pl~ loop,also referred to as i2 (Pin et al., EMBO J. 13, 342-348 (1994)). Thus it is possible e.g. to ~ Wo s6/o6l67 P~

2~ 96997 -5- - ' analyze tbe interaction of a oest compound with a ligand binding domain of a receptor of the invention using an assay for calcium ions. The chimeric receptor according to the invention can be s~ - ' by ' techniques or agents known in tbe art as ~ being suitable for, ' ,, proteins.

Aggregative derivatives are e.g. adsorption complexes vith cell ' ~ ~

In another Pmi ~ t, the present invention relates to a . . of matter ' g the hmGluR sub~pe of i-he invention.

The proteins of the invention are useful e.g. as ~ , in drug screening assays, as - ~,, fr~r ~ ,~i. and in I - methods, such as for affinity I - of a binding ligand.

A protein of the invention is obtainable from a natural source, e.g. by isolation from brain tissue, by chemical syntbesis or by rpc~m~

The invention further provides a method for preparing the hmGluR subtype of the invention, said method being . l ~. ;,. ~1 in tbat suitable host cells producing tbe receptor subtype of tbe invention are multiplied in vitro or in vivo. Preferably, the host cells are ~ ", cJ (j r J~ with a hybrid vector comprisimg an expression cassette- . ~ a promoter and a DNA scquenoe coding for said subtype which DNA is controlled by said promoter. S ' , b~, the hmGluR subtype of the inventdon may be recovered. Recovery comprises e.g. isoiating the subtype of the invendon from the host cells or isolating the host cells comprismg the subtype, e.g. from the culture broth.
P ' '.y preferred is a method for L . ' of a r ' ~Iy acdve receptor.

HmGluR muteins may be produced from a DNA encoding a hmGluR protein of the invention which DNA has been subjected to in vitro " ~ resulting e.g. in an addition, exchange andlor deletion of one or more amimo acids. For example, 1, deletional and insertional variants of a hrnGluR subtype of the invention areprepared by lC ' methods and screened for immuno-clu~li~ity with the native forms of the hrnGluR.

A protein of the invention may also be derivatized in vitro according to conventional methods known in the art.

~ ~ .

WO 96/06167 r,_l/r.. 'IQ?728 21 969q7 -6-Suitable host cells include eukaryotic cells, e.g. animal cells, plant cells and fungi, and ulic cells, such as gram-positive and gram-negative bacteria, e.g. E. coli. Preferred eukaryotic host cells are of amphibian or I -' origin.

As used herein, in vitro means ex vivo. thys including e.g. cell culture and dssue culture conditions.

This invention further covers a nucleic acid (DNA, RNA) comprising a pmified, preferably re-- -' t, nucleic acid (DNA, RNA) coding for the subtype of the invention. or a fragment of such a nucleic acid. In addition to being useful for the production of the above mentioned ' hmGluR proteins, these nucleic acids are useful as probes, thys e.g. readily enabling those skilled in the art to identify and/or isolate nucleic acid encoding a hmGluR2 protein of the invention. The nucleic acid may be unlabeled or labeled with a detectable moiety. r ~, nucleic acid according to the invention is useful e.g. im a method fom' ~ the presence of hmGluR, said method -t . ~ h~ i li~g the DNA (or RNA) encoding (or ~ ' y to) hmGluR to test sample nucleic acid and to determine the presence of hmGluR.

Purified hmGluR2 encoding nucleic acid of the invention includes nucleic acid that is free from at least one . nucleic acid with which it is ordinarily associated im the natural soyrce of hmGluR nucleic acid. Purified nucleic acid thus is present in other than in the form or setting in which it is found in natme. However, purifed hmGluR2 nucleic acid embraces hmGluR2 nucleic acid in ordinarily hmGluR expressing cells where the nucleic acid is in a ' ' location different from that of natural cells or is otherwise flanked by a different DNA sequence than that found in nature. The hmGluR2 gene maps to human ~,hll 3.

In particular, the invention provides a purified or isolated DNA molecule encoding a hmGluR2 protein of the invention, or a fragment of such DNA. By definition, such a DNA
comprises a coding single DNA, a double stranded DNA consisting of said coding DNA
and ,~ - y DNA thereto, or this . ' ~ (single stranded) DNA itself.
Preferred is a DNA coding for the above captioned preferred hmGluR2, or a fragment thereof. r ,u~ r, the invention relates to a DNA comprising a DNA coding for theabove captioned preferred hmGluR2 subtype. or a fragment thereof.

~ WOg6/06167 2 l 969q7 ~ PCr/EPssl0272s ,~

More specifically, preferred is a DNA coding for hmGluR2 or a portion thereof, p~: ' ly a DNA encoding Ihe hmGluR2 having the amino acid sequence ~l forth in SEQ ID NO:2, e.g. the DNA with the nucleotide sequence sel forth in SEQ ID NO: I .

The nucleic acid sequences provided hereim may be employed to identify DNAs encoding further hmGluR sublypes. For example, nucleic acid sequences of the invention may be used for identifying DNAs encoding further hmGlnR sublypes belonging to the samereceptor subfamily. A method for identifying such DNA comprises contacting humanDNA with a nucleic acid probe described above and identifying DNA(s) which hybridize to thal probe.

Exemplary nucleic acids of the invenlion can r' ' " ~ be ~ ;i as those nucleic acids which encode a hmGluR subtype of Ihe invenlion and hybridize lo the DNA
having Ihe sequence sel forth in SEQ ID NO.: 1, or a selected portion (fragmenl) of said DNA. Preferred are such DNA molecules encoding a hmGluR of the invendon which hybridize nnder L,~h ~ ~ conditions to Ihe sbu.~, ' DNAs.

Stringency of l.~l,.i li~iu.. refers to condilions under which pul~ ' acids hybrids are slable. Such conditions are evidenl to those of ordinary skill m the field. As known lo those of skill in the art, the stability of hybrids is reflected in the melting i , (Tm) of the hybrid which decreases ~ 1 to 15~C with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion and i , Typic~lly, the ..~: reaction is performed under conditions of higher stringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permit h~b.id;~liu.. of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68 ~C. High stringency conditions can be provided, for example, by l.~b,id;~d~ioll in an aqueous solution containing 6x SSC, 5~ Denhardl's, I % SDS (sodium dodecyl sulfate), û.l Na~
IJJ - r- , ' and 0.1 mg/ml denatured salmon sperm DNA as non specific cnmrPr~ r Following 1.~ high stringency washing may be done in several steps, with a final wash (aboul 30 min) al the h~fblid;~Lion i . im 0.2- O.lx SSC, 0.1 % SDS.

Moderate stringency refers to conditions equivalentto l~;biidi~AIiu.l in the above described solution bul al aboul 60-62 ~C. In that case the final wash is performed at the hylJlh~ r ' in lx SSC, 0.1% SDS.

Wo s6/o6l67 2 1 9 6 9 9 7 ~ o Low stringency refers to conditions equivalent to hrblhliLAIiul~ in the above described solution at about 50-52~C. In that case, the final wash is performed at the L~ idiL~ iOll ..... n.. r. in 2x SSC, 0.1% SDS.

It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g. ~ ~ -based buffers, and , Denbart's solution and SSC are well known to those of skill in the art as are other suitable hyb~hliL~ liu~ buffers (see, e.g.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbûr Laboratory Press, Cold Spring Harbor, USA, or Ausubel, F. M., et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, USA). Optimal L.r blidi~aL;U~I conditions have to be determined empirically, as the length and the GC content of the probe also play a role.

Given the g udance of the present invention, the nucleic acids of the invention are obtainable according to methods well known in the art. The present invention further relates to a process for the I , of such nucleic acids.

For ex~unple, a DNA of the invention is obtainable by chemical synthesis, by 1~ ' .DNA technology or by pol~ ~ chain reaction (PCR). Preparadon by 1. ' ~
DNA tecbnology may involve screening a suitable cDNA or genomic ]ibrary. A suitable method for preparing a DNA or of the invention may e.g. comprise the synthesis of a nutnber of -'i ' ' . their ~ . ' by PCR methods, and their splicing to give the desired DNA sequence. Suitable libraries are ~ 'ly available, e.g. the libraries employed in the Examples, or can be prepared from neural or neuronal tissue samples, e.g. i rr - orcerebellum tissue, oell lines and the like.

For an individual hmGluR subtype (and splice variants) the expression pattern in neural or neuronal tissue may vary. Thus, in order to isolate cDNA encoding a particular subtype (or splice variant), it is adv ~ to screen libraries prepared from different suitable tissues or cells. As a screening probe, there may be employed a DNA or RNA comprising ~ ' lly tbe entire coding region of hmGluR2, or a suitable ~ , ' ' probe based on said DNA. A suitable ~Ui~, ' ' probe (for screening involving Lyl .. i. I ;~ ) is a single stranded DNA or RNA that has a sequence of n~ eûti~ps that includes at least 14 contiguous bases that are the same as (or c, 11 . l AI y to) any 14 or more contiguous bases set forth in SEQ ID NO:1. The probe may be labeled with a ~ wos6/06167 2 ~ q6 9 97 . . ; ~' r g = ~

suitable chemical moiety for ready detection. The nucleic acid sequences selected as probes should be of sufficient length and ~ 1~ , so that false positive results are Preferred regions from which to construct probes include 5' and/or 3' coding sequences, sequences predicted to encode ligand binding sites, and the like. For example, either the full-length cDNA clone disclosed herein or fragments thereof can be used as probes.
Preferably, nucleic acid probes of the invention are labeled with suitable label means for ready detection upon Lyl .. ;u: ~;. . For example, a suitable label means is a radiolabel.
The preferred method of labeDing a DNA fragment is by; ~ ; .g 32P-labelled ~-dATP with the Klenow fragment of DNA pol~ in a random priming reacdon, as is weD known m the art. tl'i~ ' ' are usuaDy end-labeled with 32P-labeled ~-ATP
and pol~ ' ~ kinase. EIowever, other methods (e.g. non-radioactive) may also be used to label the fragment o m 'ig- ' ' . including e.g. enzyme labeDing and lJ;Ulill.~

After screening the library, e.g. with a portion of DNA including ! ' ' '- ~Iy the entire hmGluR2-encoding ~quence or a suitable i 'i,, ' ~ based on a portion of said DNA, positive clones are identified by detecting a hjblhl;~liu.. signal; the identified clones are . ~ .;, d by restriction enzyme mapping and/or DNA sequence analysis,and then examined, e.g. by , with the ~quences ~t forth herein, to ascertain ~vhether they include DNA encoding a complete hmGluR (i.e., if they include translation initiation and codons). E the ~lected clones are ~ , ' . they may be usedto rescreen the same or a different library tû obtain o ~ r ' 7 clones. E the library is genomic, then the u,. ~; . g clones may include e~cons and introns. If the ]ibrary is a cDNA library, then the u, ~I ~, g clones wiD include an open reading frame. In both instances, complete clones may be identified by , with the DNAs and deduced amino acid sequences provided herein.

r in order to detect any -~ l ~ of an ~ 10O. ~-- - hmGluR2 genetic screening may be carried out using a nucleotide ~quence of the invention as h.~blidi~ iU
probes. Also, based on the nucleic acid ~quences provided herein anti~n~-type thPn~p~llrir agents may be designed.

It is envisaged that the nucleic acid of the invention can be readily modified by nucleotide s~ .... nucleotide deletion. nucleotide insertion or inversion of a nucleotide stretch.

_ _ WO s6/06167 2 ~ 9 6 9 9 7 ' r~

and any cn~ nsltinn thereof. Such modified sequences can be used to produce a mutant hmGluR subtype which differs from the receptor subtypes found in nature. M~ ge"ncic may be L~ .t.. ;.. d (site-specific) or random. A mutation which is not a silent mutation must not place sequences out of reading frames and preferably will not create cr ~ Y regions that could hybddize to produce secondaty mRNA structures suchas loops or hairpins.

The cDNA or genomic DNA encoding native or mutant hmGluR of the invention can ben. ~1 into vectors for fmther I ~ r. . the invention concerns a DNA which is a hybrid vector comprising at least one of the above mentioned DNAs.

The hybdd vectors of the invendon comprise an odgin of replication or am _ 'y replicating sequence, one or more dominant marker sequences and, optionally, expression control sequences, signal sequences and additional restdction sites.

Preferably, the hybdd vector of the invention compdses an above descdbed nucleic acid insert operably linked to an expression control sequence, in particular those descdbed h~rnin~ft~r Vectors typically perform two functions in --" -' with compatible host cells. One function is to facilitate the cloning of the nucleic acid that encodes the hmGluR subtype of the invention, ie. to produce u able quantities of the nucleic acid (cloning vectors). The other function is to provide for replication and expression of the gene constructs in a suitable host, either by ~ as an e,~l-~l.l. I element or by integration into the host ~ ' - (expression vectors). A cloning vector compri~s the DNAs as descdbed above, an origin of replication or an '~, replicating sequence, ~lectable marker ~quences, and optionally, signal ~quences and additional restdction sites. An expression vector ~ compdses expression control ~quences essential for the i , and translation of the DNA of the invention. Thus an expression vector refers to a ' DNA construct, such as a plasmid, a phage, I~in~ ~
virus or other vector that, upon; ~t .~ - into a suitable host cell, results in expression of the cloned DNA. Suitable expression vectors are well known in the art and include tho~ that are replicable in eukaryotic andlor IJlu~ y~L~, cells.

Most expression vectors are capable of replication in at least one class of organisms but ~~ WO96106167 2 ~ 96997 t' r~ r = r~ 728 !~ .

can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same v ctor is transfected into ye~t or - ~ cells even though it is not capable of replicating ' . ~ 'y of the host cell .' - DNA may also be amplified by insertion into the host genome. However, the recovery of genomic DNA
encoding hmGluR is more complex than that of ~ ~g~ J replicated vector because restriction enzyme digestion is required to excise hmGluR DNA. DNA can be amplified by PCR and be directly transfected into the host cel~s without any replication c Adv _ '~" expression and cloning vector contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growtn of ' host cells grown in a selective culture medium. Host cells not 1, A, r""..
witb tbe vector contaiming the selection gene will not survive in the culture medium.
Typical selection genes encode proteins that confer resistance to antibiotics and other toxins. e.g. ampicillin, neomycin, ' or b,tn.~ u .; A-' r ~, or supply critical nutrients not available from complex media Since the ~ of the vectors is ~ u~v. ',~, done im ~Q~, an E. coli genetic marker and an _ coli origin of replication are ~lv ~ , included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript vector or a pUC plasmid.

Suitable selectable markers for ' cells are those thatenable the ~ ;r;. ,;,. . of cells competent to take up hmGluR nucleic acid, such as ~' ~d-~ ~ ' reductase (DHFR, ~Pth( ' ' resistance), thymidine kinase, or genes confering resistance to G418 or 1 ill. The cell m rr- ~ are placed under selection pressure which only those i ~ ~u are uniquely adapted to survive which have taken up and are expressing the marker.

F, ~ and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to hmGluR2 nucleic acid. Such promoter may be inducible or r -~ ,. The promoters are operably linked to DNA encoding hmGluR2 by removing the promoter from the source DNA by restticdon enzyme digestdon and in~rdng the isolated promoter sequence into the vector. Both the nadve hmGluR2 promotersequenceandmanjl~t~ulco~,~promotersmaybeusedtodirect- ..pl;r;,~,;.~..
and/or expression of hmGluR DNA. However, heterologous promoters are preferred, becau~ they generally allow for greater ~ and higher yields of expressed bmGluR2 as compared to nadve hmGluR2 promoter~

wo s6/ocl67 2 1 9 6 9 9 7 . r~

Promoters suitable for use with u-uh~uyuLiC hosts include, for example, the ~-lactamase and lactose promoter systems, alkaline r " ' a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding hmGluR, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generaUy contain a Shine-Delgarno sequence operably linked to the DNA encoding hmGluR2.

HmGluR2 gene n ~ from vectors in -' host cells may be controlled by promoters compatible with the host cell systems, e.g. promoters derived from the genomes of viruses. Suitable plasmids for expression of the hmGluR subtype of the invention in eukaryotic hostcells, p uLi~,ulally 1 cells, are e.g. ~,~X ~,~, ' ,vuu~ (CMV) promoter-containing vectors, RSV promoter-containing vectors and SV40 promoter-containing vectors and M~lTV LTR promoter-containing vectors. Depending on the nature of their regulation, promoters may be ~,u.~tiLb~i~., or n~gr~ by conditions.

T.~ ,n~ of a DNA encoding a hmGluR subtype according to the invention by higher eul~aryotes may be increased by inserting an enhancer sequence into the vector.

The various DNA segments of the vector DNA are operatively linked, i.e. they arecontiguous and placed into a functional ' ' ~ to each other.

- of vectors according to the invention employs ~,u~. -' ligation ~ . Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confrm correct sequences in the ~ J plasmids is performed in a manner known in the arL. Suitable methods for u.,Liug expression vectors, preparing in vitro transcripts, ;~ u~ C
DNA into host cells, and perforrning ~ulal~;,~ rO. assessing hmGluR expression and function are known to those skilled in the art. Gene presence, - , 1 ~ - andlor expression may be measured in a sample directly, for example, by UUII~ Southern blotting, northern blotting to quantitate the I ~ of mRNA, dot blotting (DNA or RNA analysis), in situ h~ aLiu.,, using an ~IU,UI~ ~ ' 1SI labelled probe based on a sequence provided herein. binding assays, - ~ and functional assays.
Suitable methods include those decribed in detail in the Examples. Those skilled in the art ~ wo 96/06167 2 1 9 6 9 9 7 ~ ' p~/r ~

will readily envisage how these methods may be modified, if desired.

The invention further provides host cells capable of producing the hmGluR subtype of the invention and including h~,t~. 'oO (foreign) DNA encoding said subbpe.

The nucleic acids of the invention can be expressed in a wide variety of host cells, e.g.
those mentioned above, that are r ~ or transfected with an ~ expression vector. The receptor of the imvention (or a portion thereof) may also be expressed as a fusion protein. R~ ~- cells can then be cultured under conditions whereby the protein (s) encoded by the DNA of the invention is (are) expressed.

Suitable L~lu~ yut~ include eubacteria, such as l~ ~ ..Cgal;~, or Gram-prositiveorganisms, such as E. coli, e.g. E. coli K-12 strains, DH50~ and HB 101, or Bacilli.
Further host cells suitable for hmGluR encoding vectors include eukaryotic microbes such as ~' fungi or yeast, e.g. S~,h~uu. ~ cerevisi~. Higher eukaryotic cells include insect, , ' ~ and vertebrate cells, ~ ' cells, e.g.
' ' - cell lines or fibroblas~ derived cell lines. Examples of preferred -~ cell lines are e.g. HEK 2g3 cells, CHO cells, CVI cells, BHK cells, L cells, Il~PK-1 cells, GH3 cells, and COS cells. In recent years L" ~L' ~g~n;~ of verteb}ate cells in culture (tissue culture) has become a routine procedure. The host cells referred to in this -I~L~l;. -n~r. comprise oells in in vitro culture as well as cells that are vithin a host animal.

Suitable host cells for expression of an active ' hmGluR2 adY ~ 'y e~press . ~c,, or ' G-proteins. Preferred are cells producing little, if any, ; ~C o ~ I -b~n ~p ' glutamate receptor. DNA may be stably il~ L,, ~ into the cells or may be transiently expressed according to Cull~ I methods.

Stably transfected ' cells may be prepared by t. e~ E~ cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient 1 " rr ~ I _ . ,t~ - 1 ' cells are transfected with a reporter gene to monitor i f~ ' ' efficiency.

To produce such stably or transiently transfected cells, the cells should be transfected with a sufficient amount of hmGluR-encoding nucleic acid to form hmGluR of the invention.
The precise amounts of DNA encoding hmGluR of the invention may be empirically wos6/06167 21 96997 . r~.,LI ,.~ --dPtPnninPd and optimized for a particular cell and assay.

A DNA of the invention may also be expressed in non-human transgenic animals, '~, transgenic warm-blooded animals. Methods for producing transgenic animals, including mice, rats, rabbits, sheep and pigs, are known in the art and are disclosed, for example by Hammer et aL (Nature 315, 680-683, 1985). An expression unit including a DNA of the invention coding for a hmGluR together with ~ / positioned expression control sequences, is introduced into pronuclei of fertilized eggs. T. u u ~
may be schieved, e.g. by 1l.~ ;f ' Integration of the injected DNA is detected, e.g.
by blot snslysis ûf DNA from suitable tissue sampl~s. It is preferred that the introduced DNA be ~ ' into the germ Iine of the animal so that it is passed to the animal'sprogeny.

r . a knock-out animal may be developed by . ' ~ g a mutation in the mGluR sequence, thereby generating an animal which does not express the functional mGluR2 gene anymore. Such knock-out snimal is useful e.g. for studying the role of the receptor in ' ~ , im normal and disturbed brain function.

More ~ ,ally, a knock-out animal may be developed (Le. an snimsl that does not express the f - ~-~ ~ mGluR2 gene anymore), in which one introduces a mutsted orwild-type hmGluR2 gene. Methods for producing knock-out mice are known in the srt.
The knock-out animals are useful not only for studying the role of a given " ~ .ù~
receptor, ss , ' - ' by pub]ished studies (see e.g. F. Conquet et sL, Nature 3?2, 237-243 (1994);; A. Aiba et al., Cell 79, 365-375 (1994); M. Masu et aL, Cell 80, 757-765 (1995)), but slso and, in psrticular, fûr providing a " animsl model with a suitable genetic b~l~, ' for ~ g snd expressing transgenes encoding the h( - ' aO human receptor andlor seversl of its isoforms. Expression of human receptors on a I ' ~O gene knock-out b~ ' has the unique advantsge of excluding differences in efficacies of drugs on a given receptor (in this case mGluR2) caused by species-specific sequence differences in the receptor.

Host ceDs are transfected or r ~ with the above-captioned expression or cloning vectors of this invention snd cultured in ~UII~ 1 nutrient media modified as a~U~lUIJlb~ for inducing promoters, selecting i . or amplifying the genes encoding the desired sequences. Heterologous DNA may be introduced into host cells by any method known in the art, such as ",..,~rr. ~;".. with a vectorencoding a heterologous WO 96/06167 2 1 9 6 9 9 7 - r~ 1 /~ . 1 '~728 '~:

DNA by the calcium phosphate ~v~ technique, by ele.,~l. r _ " or by Iirnf~ctin mediated. Numerous methods of t, r ~ are known to the skilled worker in the field. Successful i ' is generally recogni~ed when any indication of the operation of this vector occurs in the hos~ cell. T ~ is achieved using standard tecbniques Ip~ , to the particular host cells used.

T ~ of cloned DNA into a suitable expression vector, j r '~ of eukaryoticcells with a plasmid vector or a ~ ' of plasmid vectors, each encoding one or more distimct genes or with linear DNA, and selection of trar~fiected cells are well known in the art (see, e.g. Sambrook et aL (1989) Molecular Cloning: A T -' y Manual, Second Edition, Cold Spring Harbor Laboratory Press).

T~ or n .-- r, ." = ~I cells are cultured uslng media and culturing methods known in tbe art, preferably under conditions, whereby hmGluR encoded by the DNA is expressed.
The . , of suitable media is known to those in the art, so that they can be readily prepared. Suitable culturing media are also . ~ 'ly available.

While the DNA provided herein may be expressed in any suitable host cell, e.g. those referred to above, preferred for expression of DNA encoding functional hmGluR are eukaryotic expression systems, I ' '!/ ' expression systems, including 'ly available systems and other systems known to those of sl~ill in the art.

Human mGluR2 DNA of the invention is ligated into a vector, and introduced into suitable host cells to produce ~ ' cell llnes tbat express hmGluR2 of the invention, or specific ' of hmGluR subtypes including hmGluR2. The resulting cell llne can then be produced in amounts sufficient for -~ ' ' - qualitative and ~, ~., analysis of the effects of a receptor-specific agonist, aotagonist or allosteric modulator. AdditionaUy, mRNA may be produced by in vitro ~ " of a DNA encoding the subtype of the invention. This mRNA may be injected into Xenopus oocytes where the mRNA directs the synthesis of the active receptur subtype.
A ~I,c.L.~.,ly, the subtypc-e :~ " g DNA can be directly injected into oocytes. The transfected ' cells or injected oocytes may then be employed in an drug screening assay provided I r Such drugs are nseful in diseases associated with P~''~~ of the hmGluR subtype of the invention. Such diseases include diseases resulting from excessive action of glutamate ~ ' "y mediated by hmGluRs, such asstroke, epilepsy and chronic I. .. ,,0~ .., diseases. r, ~i~u~ useful for assessing the , , WO96tO6167 ~ ~ ~, . " I~.11~1._"''~728 2~ 96997-16-specific interaction of ~ p~ k with specific hmGluR subtypes are stabiy transfected cell lines expressing the hmGluR of the invention.

Thus host cells expressing hmGluR of the invention are useful for drug screening and it is a further object of the present invention to provide a method for identifying a compound or signal which modulates the activity of hmGluR2, said method comprising exposing cells containing h~Llulo~,w DNA encoding hmGluR of the invention, wherein said cells produce functional hmGluR2, to at least one compound or signal whose ability to modulate the activity of said hmGluR is sought to be ~ 1, and thereafter - ~ said cells for changes caused by said ' ~ Such an assay enables the ~ ~ of agonists, antagonists and allosteric modulators of the hmGluR of the invention.

In a further aspect, the invention relates to an assay for identifying C~--r ' which modulate the activity of hmGloR2, said assay . ~ ~ ~
- contacting cells expressing an active hmGluR2 and containing ~t..,.l.~g....~ DNA
encoding said hmGluR subtype with at least one compound to be tested for its ability to modulate the activity of said receptor, and - analysing cells for a difference in second messenger level or receptor activity.

In particular. the invention covers an a~say for identi~ting . ' which modulate the activity of hmGluR2, said assay . ~ ~ ~
- contacting cells expressing active hmGluR2 and contailung h. ~ DNA encoding said hmGluR subtype with at least one compound to be tested for its ability to modulate the activity of said receptor, and - ~ E, said cells for a resulting change im second messenger activity.
The result obtained in the assay is compared to an assay suitable as a negative control.

Assay methods generally require - , to variouw controls. A change in receptor activity or in ~cond messenger level is said to be induced by a test compound if such an effec~ does not occur in the absence of the test compound. An effect of a test compound on the receptor subtype of the invention is said to be mediated by said receptor if this effect is not observed in cells which do not express the recepton As used herein, a compound or signal that modulates the activity of the hmGluR of the invention refers to a compound or signal that alters the response pathway mediated by wo 96/06167 17 t ;,., ;~ 0 hmGluR2 within a cell (as compared to the absence of said hmGluR). A response pathway is activated by an ~; 1 stimulus, resulting in a change in second messenger l or enzyme activity, or resulting in a change of the activity of a .h~,..ll,.~e-bound protein, such as a receptor or ion channel. A variely of response pathways may be utilized, including for example, the adenylate cyclase response paLhway, the l ' , ' li C/ " ' calcium ion response pathway or a response pathway involving couplmg of the receptor to an ion channel. Assays to determine adenylate cyclase activity are well known in the art, and include e.g. the assay disclosed by Nakajima et aL, J. Biol. Chem. 267, 2437-2442 (1992)) Thus bmGluR2 expressing cells may be employed for the i~ of p~ ~L ' 'y low molecular weight molecules capable of acting as glutamate agonists org Preferred are low molecular weight molecules of less than 1,000 Dalton.
Witbin tbe context of the present invention, an agonist is understood to refer to a molecule tbat is capable of interacting with hmGluR2, thus mimicking the action of L-glutamate. 1 particular, a glutamate agonist is . I~ . ~. J~ ~; ,. .1 by its ability to interact with the hmGluR
of the invention, and thereby increasing or decreasing the stimulaLion of a response pathway witbin a cell. For example, an agonist increases or decreases a . ~u Ir parameter within the host cell, such as the of a second g as does the natural ligand increase or decrease said parameter. For example, in a suitable test system, wherein the hmGluR of the invention is negatively coupled to adenylate cyclase, e.g. CHO cells or BHK cells expressing hmGluR2, such an agonist is capable of ' ' g the function of hmGluR2 such that the ~ " ' , of cAMP is decreased.
~y contrast, in situations where it is desirable to tone down the activity of hmGluR2.
molecules are usefuL Witbin the context of the present invention, an antagonist is, ' ~ ~ to refer to a molecule tbat is capable of inoerac~ng with bmGluR2, but which does not stimulaoe a response pathway witbin a cell. In particular.
glutamaoe ~ are generally identified by their ability to inoeract with hmGluR2 of tbe invention, and thereby reduce the ability of tbe natural ligand to stimulaoe a response pathway within a cell, e.g. by inoerfering with the binding of ~glutamaoe to the hmGluR
of tbe invention or by inhibiting other cellular functions required for the activity of the hmGluR For example, in a suitable assay, e.g. an assay involving CHO ceLs or BHK cells e~,.~i" lg hmGluR2, a glutamaoe antagonist is capable of ~ :c ' ~ ~, the activiLy of a hmGluR of the invention such that the ability of the natural ligand to decrease the Wo 96/06167 ~ r~ 7~s 21 96~9~ -18-. f ~ cAMP un f ..~1~,;. .. is weakened. Yet another alternative to achieve an ~ '~goni~'if~ effect is to rely on o . ~ of antisense hmGluR RNA. Preferred is an agonist or antagonist selectively acting on bmGluR2. P,l d~,ul,~ly useful is an agonist or antagonist s~cill~,flly ~ n ~ c the activity of hmGluR2 without affecting the activity of any other subtype.

An allosteric modulator of a l~mGluR of the invention interacts with the receptor protein at another site than L-glutamate, thus acting as agonist or antagonist. Therefore, the screening assays decribed herein are also useful for detecting an aUosteric modulator of a receptor of the invention. For example, an allosteric modulator acting as agonist may enhance the specific interaction between the hmGluR of the invention and L-glutamate. If an allosteric modulator acts as an antagonist, it may e.g. interact with the receptor protein in such a way that bindiug of the agonist is ' - "y less effective.

An ~ ~say for a glutamate agwust or antagonist may require that dhe hmGluR of dhe invention is produced in sufficient amounts in a functional form using ~ DNA
medhods. An ~say is then designed to me~ure a functional property of the hmGluR2protein, e.g. interaction widh a ' ~ ligand. Production of the hmGluR of dheinvention is regarded ~ occurring in sufficient amounts, if activity of said receptor results in a ' '- response.

For example, ' cells, e.g. HEK293 cells, L cells, CHO-KI ceUs, LLCPK-I cells or GH3 cells (available e.g. from the American rlssue Type Culture Collection) are adapted to grow in a glutamate reduced, preferably a glutamate free, medium. A hmGluR
expression pl~mid, e.g. a pl~mid described in dhe Fxamples~ is transiendy transfected into d~ecells, e.g. by ' ,' ~,,' p ~ fA~...\ (Ausubel,F. M.,et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, USA). Cell lines stably expressing the hmGluR of the invention may be generated e.g. by li, ' "
F ~ I;n with hmGluR2 expression plasmids and a plasmid COlU~ 11.g a selectable marker gene, e.g. pSV2-Neo (Soudlcrn and Berg, J. MoL Appl. Genet. 1, 327-341 (1982)), a plasmid vector encoding dhe G-418 resistence gene. Cells surviving dhe selection are isolated and grown in the selection medium. Resistant clonal cell lines are analyzed, e.g.
for viLy widh subtype-specific antibodies or by assays for hmGluR functional responses foDowing agonist addition. Cells producing dhe desired hmGluR subtype are used in a method for detecting compounds binding to the hmGluR of the invention or in a method for identifying a glutamate agonist or antagonist ~ WO96/06167 2 1 ~ 6 9 q7 j ; ~ 728 In a further Pmho~' t, the invention provides a method for identifying ~mpo~ln~ic binding to hmGluRr', said method comprising employing the hmGluR subtype of Ihe invention in a CU~U~ binding assay. The principle underlying a ~,um,u~,Lilive binding assay is generally known in the art. Briefly, binding assays are performed by allowing the compound to be tested for its hmGluR2 bimding capability to compete with a known.
suitably labeled, ~ ligand for the binding site at the hmGluRr7 target molecule.
A suitably labeled ligand is e.g. a lad;O~Li~ labeled ligand, such as [3~]i h--- ~ , or a ligand which can be detected by its optical properties, such as ~1.$~n~ r or n..,... ,(~
After removing unbound ligand and test compound the amount of labeled ligand bound to hmGluRr' is measured. If the amount of labeled ligand is reduced in the presence of the test compound tbis compound is said to be bound to the target molecule. A ~,UlLI~LiLi~., binding assay may be performed e.g. with j r ~ or transfected host cells expressing the hmGluR of the invention or a ' cellular fraction C~ ; g teh hmGluR
of the invention.

Compound bound to the target hmGluR may modulate functional properties of hmGluR2 and may thereby be identified as a glutamate agonist or antagonist in a functional a say.

Fumctional assays are used to detect a change in the functional activity of hmGluRr' of the invention, i.e. to detect a functional response, e.g. as a result of the interaction of the compound to be tested with said bmGluR. A functional response is e.g. a change (difference) in the: of a relevant second messenger, or a change in the activity of another membrane-bound protein influenced by the receptor of the invention within cells expressing functional hmGluRr.! (as compared to a negative control). Those of skill in the art can readily identify an assay suitable for detecting a change in the level of an " ~ second messenger indicative of the expression of active hmGluR2 (functional assay). Examples include cAMP assays (see, e.g. Nakajima et aL, J. Biol.
Chem. 267, 2437-2442 (1992), cGMP assays (see, e.g. Steiner et al., J. Biol. Chem. 247, 1106-1113(1972)),~,ho;~,uh~..idylinositol(PI)turnoverassays(Nakajimaetal.,J.Biol.
Chem. 267, 2437-2442 (1992)), calcium ion flux assays (Ito et al., J. Nu~u,h~ll. 56, 531-540 (1991)), ~ ,.. h ~ acid relea~e as ays (see, e.g. Felder et al., J. Biol. Chem.
264, 20356-20362 (1989~), and the lLke.

More specifically, according to the invention a method for detecting a glutamate agonist comprises the steps of (a) exposing a compound to the hmGluR subtype of the invention wo 96/06167 2 ~ q 6 q q 7 r coupled to a respon~ pathway, under condidons and for a time sufficient to allowinteraction of the compound with the receptor and an associated response through the pathway, and (b) detecdng an increase or decrease in the sdmuladon of the response pathway resulting from the interacdon of the compound with hmGluR2, reladve to the absence of the tested compound and therefrom '~ ~ the presence of a glutamate agonisL

A method for idendfying a glutamate antagonist compri~s the steps of (a) exposing a compound in the pre~nce of a known glutamate agonist to nmGluR2 coupled to a respon~ pathway, under condidons and for a time sufficient to allow interacdon of the agonist with the receptor and an associated respon~ through the pathway, and (b)detecdng an inhibition of the sdmulation of the respon~ pathway induced by the agonisL
satd inhibition resuldng from the interacdon of the compound with htnGluR2, reladve to the ' of the respon~ pathway by the glutamate agonist alone and therefrom the pre~nce of a glutamate antagonisL Inhibitdon may be deoecoed, e.g. if the test compound compeoes with the glutamate agonist for hmGluR2. C- . ' which may be screened udlizing such method include blocking andbodies ~;I.~U~ binding to hmGluR2. 1' Ih.... --- ..e such an assay is useful for the screening for ~ , ' mteracdng v~ith L ,. ' - - In this ca~, the agonistic effect is neutralized or reduced, e.g. by binding of the oest compound to the agonisL thus affecting agonist interacdon with the receptor. Examples are soluble hmGluR fragments comprlsing part or all of the ligand binding domain.

r ~ ~ly, inoeracdon of an agonist or antagonist with hmGluR2 of the invendon denotes binding of the agonist or antagonist to said hmGluR.

As employed herein, condidons and dmes sufficient for interaction of a glutamaLe agonist or antagonist candidaoe with the receptor wiU vary with the source of the receptor, however. condidons generally suitable for binding occur between about 4~C and about 40~C, preferably between about 4~C and about 37~C, in a buffer soludon between 0 and 2 M NaCI, preferably between 0 and 0.9 M NaCI, with 0.1 M NaCI being ~u~Liuul~ly preferred, and witnin a pH range of between 5 and g, preferably between 6.5 and 8.
Sufficient dme for the binding and respon~ will generaUy be between about 1 ms and about 24 h after exposure.

Within one emho~iim~n~ of the present invention, the response pathway is a ~ W0 96/06167 r~ 728 2 1 ~997 ~ ~ t r mPml -bound adenylate cyclase pathway, and, for an agonist, the step of deoecangcomprises measuring a reduction or incre~e, preferably a reduction, in cAMP producaon by the membrane-bound adenylaoe cycl~e response pathway, relaave to the cAMP
producaon in the relevant control setup. For the purpo~ of the present invention, it is preferred that the reductaon or increase in cAMP producaon be equivalent or greaoer than tbe reduction or incre~e induced by ~glutamaoe applied at a ~ c, r " g to its IC50 value. For an antagonist, tbe soep of detecting comprises measuring in the presence of the antagonist a smaller ~glutamate induced decrease or incre~e in cAMP
production by the l ' -bound adenylate cycl~e response pathway, as compared to the cAMP producaon in the absence of the antagonist. The of cAMP may beperformed afoer cell destrucaon or by a cAMP sensiave molecular probe loaded into the cell, such as a A ' dye, which changes its properaes, e.g. its fl~ rPcrPnt properties, upon bmding of cAMP.

Cyclic AMP productaon may be me~ured using methods weU known in the art, including for instance, the method described by Nakajima et al., supra, or using ~ "~, available kits, e.g. kits comprising ~ .. L ~1 cAMP, e.g. [l251lcAMP or [3HlcAMP.
Exemplary kits are the S~int~ tlnn Proximity Assay Kit by Amersham, which measures the producaon of cAMP by . , of iodinaoed-cAMP with cAMP anabodies, or the Cyclic AMP [3Hl Assay Kit by Amersham.

In assay systems using cells expressing hmGluR2 that is negaavely coupled to theadenylate cycl~e pathway, i.e. which cause a decre~e in cAMP upon ! " ' " and anincrease in cAMP upon reduction of ' it is preferred to e~cpose the cells to a compound which reversibly or i~ stimulaoes the adenylate cyclase, e.g. forskolin, orwhichisa~' .' ' inhibitor,such~ ~~L ~IL~ y~ (IBMX),prior to addition of the (pooenaal) receptor agonist or antagonist.

Within another ' ~ ' of the invenaon, the response pathway is the Pl hydrolysislCa2+ -~ '1i7~tinn pathway. Such an ~say for c' ~ the specific inoeracaon of a test compoumd with the hmGluR subtype of the invenaon may be r '' 1~ linked to changes in the ~ calcium ion (Ca2+) . Several methods for a change in the " ' .u~ ~ u,-'i~ of Ca2+ are known in the art, e.g. a method involving a calcium ion sensiave fluoroscent dye, such as fura-2 (see Cryl.Lcw;~
et al., J. Biol. Chem. 260, 3440-3450, 1985), fluo-3 or Indo- 1, such as the calcium fluor QuinZ method describe by Chaiest et al. (J. Biol. Chem. 259, 8679-8773 (1993)), or the WO96/06167 I~ N'77_8 - 2? _ - ~

aequorin ~ ' , ut~i.. method described by Nakajima-Shimada (Proc. Natl Acad. Sci.
USA 88, 6878-6882 (1991)). In one; l - ' of the invemion, intr~rP~ . calciumionc ismeasuredby , A y in ' cells loaded with calcium sensitive A ' dyes fluo-3 or fura-2. These may be performed using cells grown in a coverslip allowing the use of an inverted ll~il,l ui,~u~,e and video-imaging ~ lr,g; .5 or a n, ... -~f - p~ : to measure calcium - at the single cell level. For both ~prrn~~hPs, cells j r ~ with a hmGluR2 expressing plasmid have to be loaded with the calcium indicator. Tû this end, the growth medium is removed from the cells and replaced wihh a solution containing fura-2 or fluo-3. The cells are used for calcium I r '1~, during the following 8h. The IlP.~,Lunu~lul~l~,h~ follows standard ~

.Ca2f signals resulting from functional interac~rion of ~ r ~ with tbe target molecule can be transient if the compound is applied for a limited time period, e.g. via a perfusion system. Usmg transient application several can be made with the same cells allowing for internal controls and high numbers of . , ~ tested.

Functional coupling of the hmGluR of the invention to Ca2+ signaling may be achieved, e.g. in CHO cells, by various methods:
(i) Co ~ of a ' ~ hmGluR of the invention and a ' cation charmel, acavity of which is r ~ly linked to the activity of the hmGluRr';
(ii) e cpression of a chimeric hmGluR receptor, which directly shmulates hhe PI/Ca pathway;
(iii) c(~.l~ of ... ~..-.h -- -' hmGluR of the invention with a rPrr,ml -Ca2~-permeable cAMP dependent cation channel.

In other expression systems fimctional coupling of hmGluR2 to Ca2 ' signalling may be achieved by j r ~ of the hmGluR of the invention if these cells natu~ally express (i) voltage gated Ca charmels, activity of which is functionaUy linked to activity of mGluRs or (ii) Ca2+-permeable cAMP dependent ion channels. For example, GH3 cells whichnaturally express vollage-gated Ca channels, directly allow application of Ca2~ assays to test for hmGluR2 functional achvity by ~ul~ r~, n ., n of hmGluRs.

Further cell-based screening assays can be designed e.g. by UUII~UUUIulg cell lines in which the expression of a reporter protein. i.e. an easily assayable protein, such as ~ WO96/06167 2 1 9 6 9 9 7 ~ t I~.~

~_g~ f .r~- (CAT) or luciferase, is dependen~ on tbe function of a hmGluR of the invention. For example, a DNA construct comprising a cAMP response element is operably linked to a DNA encoding luciferase. The resulting DNA construct . g the enzyme DNA is stably transfected into a host cell. The host cell is tnen transfected with a second DNA construct containing a ftrst DNA segment encoding the hmGluR of the invention operably linked to addidonal DNA segments necessary for the expression of the receptor. For example, if bindrng of an agonist to the hmGluR of the invention results in decreased cAMP levels, tbe expression of luciferase is induced or decreases, do.~ ' " E, on the promoter chosen. The luciferase is exposed to luciferin, and tbe photons emitted during oxidation of luciferin by the luciferase is measured.

The drug screening assays provided herein will enable ' - and design of receptor subtype-specific . ' . r ' ~ Iigands binding to hmGluR2, eventually leading to the d~ r of a disease-specific drug. If designed for a very specific interaction with only one pardcular hmGluR subtype (or a I ' ' selecdon of lunGluR subtypes) such a drug is mosL likely to exhibit fe ver un vanted side effects than a drug identified by screening with cells that express a(n) (unlcnown) variety of receptor subtypes. Also, testing of the smgle receptor subtype of the invention or specific ' ~ of different receptor subtypes witb a variety of potential agonists or ,, ~ provides additional - r ' with respect to the function and activity of individual hmGluR2 protein and should lead to the ' - and design of that are capable of very specific interaction with one or more receptor subtypes.

In anotner: b~ ' tne invention provides polyclonal and - ~ 1 antibodies generated against hmGluR2. Such antibodies may useful e.g. for ~, including ~ ' ' ~ as well as diagnostic and tberapeutic ~l~p~ ;, c For example, antibodies specifc for tbe L 1- ~ domain, orporlions thereof, of hmGluR2 can be applied for blocking the i ' O hmGluR subtype.

The antibodies of the invention can be prepared according to methods well known in the art using as antigen the hmGluR of the invention, a fragment tbereof or a cell expressing said subtype or fragment. The antigen may represent the active or inactive form of the receptor of the invention. Antibodies may be capable of 1' ., ' ,, between the active or inactive form. Factors to consider in selecting subtype fragments as antigens (either as _ _ _ _ . . .. . .. . ... . . . .. . .. .. .. .. . . . . . _ . . . _ wo 96/06167 2 1 9 6 9 9 7 P ~ "~ ~ 7728 synthetic peptide or as fusion protein) include ~ " dC~,~D;,ibil;~y (i.e. extracellular and ~ u,ul~l~;c domains) and uniqueness to the particular subtype.

PalL~,ul~ly useful are antibodies selectively ~ g and binding to hmGluR2. The antibodies of the invention can be ' ' to a subject in need thereof employing standard methods. One of skill in the art can readily determine dose forms, treatment regimens etc, depending on the mode of - ' employed.

The invention II~uL~,ukuly relates to the specific; ' ' as described in the Examples which serve to illustrate the present invention but should not be construed as a limitation thereof.

Ahl~ h~Liù..s. hmGluR = human ll..,~b~J~.u~,;., glutarnate receptor, . ~ ---- '. .,li.8 ~mDIe 1: Clonin~ and ex~ression of cDNA encodint, hmGluR2 1.1 cDNA ~' ~ p. Lu~ hurnan brain N-terminal and C-terrninal fragments of the rat mGluR2 cDNA (fragment nt lg2 to 518 and fragment nt 1983- 2810. Tanabe et Pl . Neuron ~, 169-179 (1992)) are generated by PCR from single stranded rat forebrain cDNA.

Single strand cDNA synthesis is carried out with 1,ug rat forebrain poly(A)+ RNA, 80 U
M-MLV reverse , (BRL), 25 mM Tris-HCI pH 8.3, 37.5 mM KCI. 1.5 mM
MgC12. 10 mM ~ - ' 1, 1 mM each dATP. dCTP, dGTP, dl'rP, 50 mglml oligo-dTl2-l8 (r ~ ), and 2 U RNAsin (Promega) at 37~C for 60 min. The 5 and3- primers used for PCR are ATGGAATCACT~iCTTGGGlT/TGAGGCAGG-CACAAAGTCCA for the N-terminal fragment ~ ' ' 192 to 518) and GTCAAG-GCTTCCGGTCGGGA/'I~AAAGCGACGAC~ l WA for the C-terminal fragment x - ' 1983 to 2810). l~,L~Iy. PCR reactions are performed using the GeneAMP DNA ~ , kit (Perkin Elmer Cetus) under the following conditions:
93~C for 0.5 min, 56~ C for 1.5 min, and 72~C for 3 min for 40 cycles. The amplifled DNAs are gel purified. cloned into the Smal site of pRl , SK. and .~ .; d by DNA -- I - g (Sequenase T7 llol.~ Kit, United States ~- ~ ~' '~).
2x 106 plaques of human fetal brain and human adult I . r ~ ~ cDNA libraries.
t~ ;i in Lambda-ZAPII (Stratagene) from oligo-(dT) and randomly primed poly(A)+ RNA, are screened --, 'Iy with N- and C-terminal rat mGluR2 probes.
M',-~l)ULIU~iC GluR2 probes are generated by random priming of gel purified fragments WO 96/06167 2 1 ~ 6 9 9 7 P~ ~ 7728 r~ c using [o~-32P]dCTP. H~l., ;.li, -I;nA~ are carried out overnight at 60~ C in Sx SSC/Sx Denhardt'slS0 mM Na2HP04/10 mM EDTA/1% SDS/50 ~Lg/ml Herring Testis DNA/20 ,ug/ml yeast RNA. Washes are done for 30 min each at 25 C in Sx SSC/0.2% SDS, 2xSSC/0.2% SDS, and lx SSC/0.2% SDS. Five plaques h~l ' e to the N- and C-terminalrat mGluR2 fragments are purified by a second and third round of screening and five cDNA inserts are rescued into Bluescript SK phagemids by in vivo excision. cDNA
inserts are . ~ d by restriction enzyme mapping and DNA ~ ~ The cDNA
clones show restnction maps, which differed only at the very 5' and 3' end. The largest cDNA clone, hmGluR2.1, contains a 4.1 kb Kpnl/Notl fragment. The entire coding sequence is sequenced on both strands. The DNA sequence coding for the hmGluR2 protein and the deduced amino acid sequence are set forth in SEQ ID NOs. I and 2, 1.2 t'n ~ of hrnGluR2 e~cpression construct and expression in ' cells The 4.1 kb insert of the cDNA I ~ "'7 1 iS cloned du .. of the mouse CMV
promoter in the ' expression vector pSMC (Asselbergs and Grand, 1993) in~o the ' ' c..~d NotVKpnl sites resulting in the expression construct p.~M~I ~lnR~
Chinese hamster ovary cells (CHO-K1) are r ~ with p~M~' r~ r7C and pSV2-Neo (Southern and Berg, Journal of Molecular and Applied Geneacs 1, 327-341(1982)) using lipofecan mediated gene truAsfer (Gibco-BRL). Thirty-two G-418 resistant clonal cell lines are isolated and analyzed for mGluR2 protein expression by ~ ~;L~ with an ~ antibody ( ~ see infra) and functional responses follo ving agonist addiaon via cAMP ~- ' assay (see infra).

ExamDle 2: T ~1 ~ of hmGluR2 Drotein exDression with subtYDe-specific bmGluR anabodies llmGluR2 expression is analyzed by ~ .~h~ hy with subtype-specific hmGluR anabodies (see Example 5). 1 to 3 days after j r '- cells are washed twice with phosphate buffered saline (PBS), fD~ed with PBS/4% ~ ~r~," Pl i h~d~; for 10 min and washed with PBS. Cells are ~ ' " ' with PBS/0.4 % Triton X-lO0, followed by washing with PBS/10 mM glycine, and PBS. Cells are blocked with PBSTB (lx PBS/0.1% Triton X-100/1 % BSA) for I h md ~ y incubated with ' hmGluR anasentm (0.5 - 2.0 ilg/ml in PBSTB) for l h. After three washes with PBS, cells are incubated for 1 h with alkaline peroxidase conjugated goal anri-rabbit IgG (1:200 in PBSTB; Jackson Immuno Re~arch). Cells are washed three ......... . _ .... . .. .. .. .. .. _ . . ... _ . _ . .... _ . .... . . .

wo s6/06l67 ~ ' r~ "~ 1~
2~96997 26 times with PBS and vi~y is detected with 0.4 mgfm~ p ~ r (Biorad)/1 mg/ml F~t Red (Biorad)/10 mM Levamisole (Sigma)/100 mM Tris/HCI pH
8.8/100 mM NaCI/50 mM MgC12. The staining reaction is stopped after 15 min by ' washing with PBS. Four cell lines, each I ~ expressing hmGluR2 are identified by ExamPle 3: Use of stable cell lines ex~ressin~ hmGluR2 for the screenin~ of modulators of receDtor activitv Stable cell lines expressing hmGluR2 are nsed to screen for agonists, antagonists and allosteric - ~ ~ Such , ~ are identified by binding studies employing t3H]glutamate and/or . -- I.f \~ - of changes in ~I--lor second messenger levels (tCAMP]. [Ca2+]).

3.1 cAMP ~~ ' ~ ~ y Ligand binding and a" ~ ~ ~ depression of forskolin stimulated cAMP
a ' (changes in the '' ' cAMP . ) are analyzed by cAMP
' 1~ (A ~ ). Cells are seeded in 12-well plates at a density of 0.5-2.0 x 105 cells per well and grown for 2 to 4 days until a confluent layer of cells is obtained.
Cells are w~hed twice with PBS and incubated for 20 min in PBS containing 1 mM
3-isobutyl-1 f- - aBMX). Cells are imcubated with fresh PBS containing 10 IIM forskolin, I mM IBMX and a known hmGluR agonist for 20 min. The agonistic effect is stopped and cAMP produced by the cells is released by adding 1 ml of ' .. . -HCl mix (100 ml of ethanol, 50 ml of water, 1 ml of 1 M HCI) after having ~pirated the drug containing medium. cAMP levels are determined by a cAMP
r~ y involving [3H] cAMP (Amersham).

HmGluR2 is negatively coupled to adenylate cycl~e when expressed in CEIO cells.
Agonist binding leads to an inhibition of forskolin imduced cAMP ' The rank order of agonist potencies is f~2S,3S,4S)-~-(~,~bu~y~ ,lu~lul~jl)-glycine >
(lS,3R)-1 .~lu~,yl~ p .: r-l.3-d;c~bw~yl;c acid = ~glutamate >

3.2 ~ ~- - of ~ r ~ ~r tCa2+]
Cells I r ~ with a hmGluR2 expression pl~mid, e.g. the above expression pl~mid, are loaded with a calcium sensitive fluorescent dye such as fura-2 or fluro-3. To achieve tms cells are plated in single wells, single wells containing a coverslip, or 96-well plates and grown for I to 5 days until a 50-100 ~o confluent layer of cells is obtained. Wells are ~! WO 96/06167 ~ h washed three times with a balance salt solution (BBS) and incubated for lh in BBS
followed by three additional washings with BBS. Then cells are incubated for 20 ~o 60 rr~in in a solution containing 50 ~Lg fura-2-AM (or fluro3-AM) (Molecular Probes, Inc.) 4.99 ml BBS, 75 ~I DMSO and 6.25,ug Pluronic (Molecular Probes, Inc). The cells are washed 3 times with BBS containing 2 mg/ml bovine albumin followed by three washes in BBS. After allowing recovery of the cells for at least 10 min they are used for Illi ~lULIUUl~ ' ~ ' of tCa2+l.

Cells are transferred to an apparatus for fluometry such as an inverted ~ IUD~UYC, a ~1~ m ~ of a n~ reader. r of the calcium indicator (e.g.
fura-2 or fluo-3) is induced by " ' with light of a wavelength covered by the excitation spectrum of the dye (fura-2: 340/380 nm, fluo-3 480 nm)~ An increase in " ' free calcium ion ~ is monitored as an increase of fura-2 or fluro-3 excited at 340 nm and 480 mm, I~ ,IY~ or a decrease of fura-2 r~ e~tcited at 380 mm.
As a positive control, ~glutamate is applied at a, ~ g to its EC50 value onto the cells, thereby inducing a ' ' increase in the ' ' calcium ion ~ ~ A test compound is said to be an agonist if it induces a Ca2+ signal ' ' to that induced by glutaunate. A test compound is said to be an antagonist if the glutamate induced calcium signal is smaller in the presence of the test compound than in the absence of the test compound.

E~amDle 4: Chimeric hmGluR2 r ~ ~ domains of mGluRl, y~u~ 1~, the second ~ " ' loop (i2) and the C-terminal region, have been shown to be critical for binding of G-proteins, which activate the I ' - "~ ', ClCa2+ signaling pathway, without changing the P~ e; ~I profle of the receptor (Pin et al., EMBO J. 13, 342-348, (1994)~.
PCR ~ ~ techniques are used to exchange ~ ' '- domains of hmGluRs 2 with . - - - ~r - ~ g domains of hmGluRI. Stable CHO cell lines are generated with hmGluR2/1 chimeric expression constructs allowing to analyze the influence of ~d ' of receptor activity (hmGluR2) using Ca2+-dependent assays.
(i) The cDNA clone hmGluR2.1 is used forthe . Ul~livu of chimera (ii) The ~.. ~... 1.. r ~ region of hmGluR1 is cloned by PCR using primers derived from Masu et al., 1991, supra The cliV '~ ' with the sequence 5 -TATCTTGAGTGGAGTGACATAG-3' g to nt 1753 to 1774 of the Masu sequence) is used as sense primer. The WO 96/06167 2 1 9 6 q 9 7 ; ~ PCT/EP9Y02728 antisense primer has the sequence 5'-ACTGCGGAC(~ f~ l CAGG-3' ~.u--, r ' g to nt 2524 to 2544 of the Masu sequence. The C-terminal end of splice variants la, lb and lc is cleaved by PCR using primers derived from Masu et al., 1991, Tanabe et aL, 1992, supra, and Pin et al. (Proc. Natl. Acad. Sci, USA, 89, 10331-10335 (1992)), .~,Div~,~,f~ . The ~ ' having the sequence 5 '-AAACCTGAGAGGAACGTCCGCAG-3 ' (1- -- -r '' g to nt 2521 to nt 2543 of the Masu sequence) is used as ~nse primer. The ~1 "~ u~ having the sequences 5'-CTACAGGGTGGAAGAGClTTGCTr-3' 1 . ' ~ to nt 3577 to 3600 of the Masu sequence, 5'-TCAAAGCTGCGCATGTGCCGACGG-3H,~ r " v to nt 2698 to 2721 of the Tanabe sequence, and 5'-TCAATAGACA~J1~ 111 1~iGCGGTC-3' L ~ ~ to nt 2671 to 2694 of the Pin sequence are used as antisense primers for l ~' '' 1 ~ lb and lc, l~D~
Tbe PCR fragment is cloned into pR' .: II and sequenced completely.
(iii) A chimeric cDNA fragment wherein the i2-loop of hmGluR2 (nt 1966-2037 of SEQ
ID NO:l) is replaced with the cul.. r " ~ sequences of hmGluRl is generated by PCR
(as described in Pm e~ al., 1994, supra). The fragment ist digested v,~ith Bsu361 and Dram which cut at unique restriction sites flanking the i2-loop. The chimeric Bsu36I/Dram fragment is exchanged for the Bsu36I/Dram fragment of clone hmGluR~ 1.
(iv) Additional -~r - of the C-terminal domain of hmGluR2 with the . r ~
~quences of the above mentioned hmGluRI splice variants is achieved by using theunique restriction sites Dram and KpnI flanking the C-terminal end of hmGluR2.
(v) The resulting chimeric hmGluR21hmGluRI cDNA is sequenced and digested with KpnI and NotI, thereby releasing the complete cDNA from p~ p~ For stable expression in CHO ceDs, the chimeric cDNA is ' ' t-c..duJ and cloned into blunt-ended NotI site of the -' expression vector pCMV-T7-2 for stable expression of chimeric hmGluR2/1 receptor in CHO ceLs.

Example 5: Generation and aPplication of anti-hmGluR2 antibodies Peptides cu... r ' Ig to the deduced C-terminal amino acid sequences of hmGluR2 are D,~ ..th~;l and coupled to ovalbumin or Tentagel. Polyclonal antisera are raised in rabbits. Human mGluR2 specific antibodies are purified from the antisera by ~nr~ffif if y ~Li~ on peptide columns. The hmGluR2 specific antibodies are .. 1, - ,.. ~ ;1 by EirlSA and 8 with v ~-S-t~ rti~/hmGluR

~ WO 96/06167 r~ D
2 1 9 6 9 9 7 L,; ' fusion prooeins (produced in ~ coli) or human brain extracts. Antibodies specilic for hmGluR2 are used to detect hmGluR2 receptos in transfected cells and to analyze the cellular and subcellular expression pattem of the hmGluR2 proteins in tissue sections of human brain mater}~l.

Antibodies are raised against different hmGluR2-specific peptides consisting of 20 amino acids and fusion proteins expressed in E.coli. Peptides are ;~J ' ' by solid-phase synthesis, coupled to keyhole limpit l.e..lo~"~ (KLH) or ovalbumin with oluh~ ch~d~,. PCR fragments containing the entire putative i~ P~ C-terminal fragment of hmGluR2 are cloned as BamHVEcoRI fragments into the E. coli expression plasmid pGEX-2T (Guan and Dixon, Analytical B ' y 192. 262-267 (1991)) generating g' ' -S-transferase(GST)/hmGluR fusion genes. E. coli DH5a cells (Gibco-BRL) carrying expression plasmids with GST/hmGluR fusion genes are grown ovemightat 37~C in LB medium/100 mg/ml ampicillin. The cultures are diluted 1:30 in LB and grown for 2 h at 30~C Expression of fusion proteins is induced by treatment with 0.1 mM isopropyl-b-D 7~ ' X r.~l os.dc for 3 h at 30~C Cells are harvested by r ,, ' at 5,000 x g. The fusion protein is isolated using glutathione affinity WO 96106167 1 ~
21 q6997 '~ - ' ~U~N~ LISTING

(1) GENERAL INFORNATION:

(i) APPLICANT: :
(A) NA~E: CIBA-GEIGY AG
(B) STREET: Klybeckstr. 141 (C) CITY: Basel (E) COUNTRY: SCHWEIZ
(F) POSTAL CODE (ZIP): 4002 (G) TELEPHONE: +41 61 69 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991 (ii) TITLE OF INVENTION: Glutamate Receptor (iii) NUMBER OF ~U~N~:S: 2 (iv) COMPUTER R~nARn~ 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 CXARACTERISTICS:
(A) LENGTH: 2618 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linea~ -(ii) MOLECULE TYPE: cDNA

RECTIFIED SHEET (RULE 9~) ISA/EP

WO96/06167 2 1 q 6 9 9 ~ . P ./~ 7~ ~728 (ix) FEATURE:
~A~ NAME/KEY: CDS
(B) LOCATION: l..26l8 (D) OTHER INFORMATION: /product= "hmGluR2 (Xi ) ~ ~N~ DESCRIPTION: SEQ ID NO: l:

Met Gly Ser Leu Leu Ala Leu Leu Ala Leu Leu Pro Leu Trp Gly Ala l 5 l0 15 GTG GCT GAG GGC CCA GCC AAG AAG GTG CTG ACC CTG GAG GGA GAC TTG
Val Ala Glu Gly Pro Ala Lys Lys Val Leu Thr Leu Glu Gly Asp Leu GTG CTG GGT GGG CTG TTC CCA GTG CAC CAG AAG GGC GGC CCA GCA GAG
Val Leu Gly Gly Leu Phe Pro val His Gln Lys Gly Gly Pro Ala Glu GAC TGT GGT CCT GTC AAT GAG CAC CGT GGC ATC CAG CGC CTG GAG GCC
Asp Cys Gly Pro Val Asn Glu His Arg Gly Ile Gln Arg Leu Glu Ala ATG CTT TTT GCA CTG GAC CGC ATC AAC CGT GAC CCG CAC CTG CTG CCT
Met Leu Phe Ala Leu Asp Arg Ile Asn Arg Asp Pro His Leu Leu Pro GGC GTG CGC CTG GGT GCA CAC ATC CTC GAC AGT TGC TCC AAG GAC ACA
Gly Val Arg Leu Gly Ala His Ile Leu Asp Ser Cys Ser Lys Asp Thr R~CTIFIED SHEE~ (RULE g1) IS~JEp WO96~06167 ~ P~ . IQ7728 His Ala Leu Glu Gln Ala Leu Asp Phe Val Arg ~la Ser Leu Ser Arg Gly Ala Asp Gly Ser Arg His Ile Cys Pro Asp Gly Ser Tyr Ala Thr His Gly Asp Ala Pro Thr Ala Ile Thr Gly Val Ile Gly Gly Ser Tyr AGT GAT GTC TCC ATC CAG GTG GCC AAC CTC TTG AGG CTA TrT CAG ATC 480 Ser Asp Val Ser Ile Gln Val Ala Asn Leu Leu Arg Leu Phe Gln Ile Pro Gln Ile Ser Tyr Ala Ser Thr Ser Ala Lys Leu Ser Asp Lys Ser Arg Tyr Asp Tyr Phe Ala Arg Thr Val Pro Pro Asp Phe Phe Gln Ala Lys Ala Met Ala Glu Ile Leu Arg Phe Phe Asn Trp Thr Tyr Val Ser Thr Glu Ala Ser Glu Gly Asp Tyr Gly Glu Thr Gly Ile Glu Ala Phe Glu Leu Glu Ala Arg Ala Arg Asn Ile Cys Val AIa Thr Ser Glu Lys ~ WO96/06167 2 ~ 96997 P~

Val Gly Arg Ala Met Ser Arg Ala Ala Phe Glu Gly Val Val Arg Ala Leu Leu Gln Lys Pro Ser Ala Arg Val Ala Val Leu Phe Thr Ar~ Ser Glu AS~ Ala Arg Glu Leu Leu Ala Ala Ser Gln Arg Leu Asn Ala Ser Phe Thr Trp Val Ala Ser Asp Gly Trp Gly Ala Leu Glu Ser Val Val Ala Gly Ser Glu Gly Ala Ala Glu Gly Ala Ile Thr Ile Glu Leu Ala 305 . 310 315 320 Ser Tyr Pro Ile Ser Asp Phe Ala Ser Tyr Phe Gln Ser Leu Asp Pro Trp Asn Asn Ser Arg Asn Pro Trp Phe Ar~ Glu Phe Trp Glu Gln Ar~

Phe Arg Cys Ser Phe Arg Gln Arg Asp Cys Ala Ala ~is Ser Leu Ar~

Ala Val Pro Phe Glu Gln Glu Ser Lys Ile ~et Phe Val Val Asn Ala WO 96106167 2 1 9 6 9 9 7 . I P~~ ~"28 Val Tyr Ala Met Ala His Ala Leu His Asn Met His Arg Ala Leu Cys Pro Asn Thr Thr Arg Leu Cys Asp Ala Met Arg Pro Val Asn Gly Arg CGC CTC TAC AAG GAC TTT GTG CTC AAC GTC AAG m GAT GCC CCC m 1296 Arçr Leu Tyr Lys Asp Phe Val Leu Asn Val Lys Phe Asp Ala Pro Phe CGC CCA GCT GAC ACC CAC AAT GAG GTC CGC ~rTT GAC CGC TTT GGT GAT 1344 Arg Pro Ala Asp Thr ~is Asn Glu Val Arg Phe Asp Arg Phe Gly Asp GGT ATT GGC CGC TAC AAC ATC TTC ACC TAT CTG CGT GCA GGC AGT: GGG 1392 Gly Ile Gly Arg Tyr Asn Ile Phe Thr Tyr Leu Arg Ala Gly Ser Gly Arg Tyr Ar~ Tyr Gln Lys Val Gly Tyr Trp Ala Glu Gly Leu Thr Leu Asp Thr Ser Leu Ile Pro Trp Ala Ser Pro Ser Ala Gly Pro Leu Ala GCC TCT CGC TGC AGT GAG CCC TGC CTC CAG AaT GAG GTG AAG AGT_GTG 1536 Ala Ser Arg Cys Ser Glu Pro Cys Leu Gln Asn Glu Val Lys Ser Val CAG CCG GGC GAA GTC TGC TGC TGG C~C TGC ATT CCG TGC CAG CCC TAT 1584 Gln Pro Gly Glu Val Cys Cys Trp Leu Cys Ile Pro Cys Gln Pro Tyr ~ WO96/06167 2 1 969~7~ P~ 728 Glu Tyr Arg Leu ASp Glu Phe Thr Cys Ala ASp Cys Gly Leu Gly Tyr 530 535 , 540 Trp Pro Asn Ala Ser Leu Thr Gly Cys Phe Glu Leu Pro Gln Glu Tyr Ile Arg Trp Gly Asp Ala Trp Ala Val Gly Pro Val Thr Ile Ala Cy8 CTC GGT GCC CTG GCC ACC CTG m GTG CTG GGT GTC TTT GTG CGG CAC 1776 Leu Gly Ala Leu Ala Thr Leu Phe Val Leu Gly Val Phe Val Arg His Asn Ala Thr Pro Val Val Lys Ala Ser Gly Ar~ Glu Leu Cys Tyr Ile Leu Leu Gly Gly Val Phe Leu Cys Tyr Cys Met Thr Phe Ile Phe Ile Ala Lys Pro Ser Thr Ala Val Cys Thr Leu Arg Arg Leu Gly Leu Gly Thr Ala Phe Ser Val Cys Tyr Ser Ala Leu Leu Thr Lys Thr Asn Arç~

6~5 650 655 .=

Ile Ala Arg Ile Phe Gly Gly Ala Arg Glu Gly Ala Gln ArsJ Pro Arg 7 2 ! 9 6 9 9 7' - ' ~ ' ~ r ~

-36- _ Phe Ile Ser Pro Ala Ser Gln Val Ala Ile Cys Leu Ala Leu Ile Ser Gly Gln Leu Leu Ile Val Val Ala Trp Leu Val Val Glu Ala Pro Gly Thr Gly Lys Glu Thr Ala Pro Glu Arg Arg Glu Val Val Thr Leu Arg Cys Asn HiS Arg Asp Ala Ser Met Leu Gly Ser Leu Ala Tyr Asn Val Leu Leu Ile Ala Leu Cys Thr Leu Tyr Ala Phe Asn Thr Arg Lys Cys CCC GAA AAC TTC A~C GAG GCC AAG TTC ATT GGC TTC ACC ATG TAC ACC 2304 Pro Glu Asn Phe Asn Glu Ala Lys Phe Ile Gly Phe Thr Met Tyr Thr Thr Cys Ile Ile Trp Leu Ala Leu Leu Pro Ile Phe Tyr Val Thr Ser Ser Asp Tyr Arg Val Gl~ Thr Thr Thr Met CYs Val Ser Val Ser Leu Ser Gly Ser Val Val Leu Gly Cys Leu Phe Ala Pro Lys Leu His Ile 21 9~997 W O96/06167 P~ 728 ATC CTC TTC CAG CCG CA& AAG AAC GTG GTT AGC CAC CGG GCA CCC ACC 2496 Ile Leu Phe Gln Pro Gln Lys Asn Val Val Ser His Arg Ala Pro Thr AGC CGC TTT GGC ~GT GCT GCT GCC AGG GCC AGC TCC AGC CTT GGC CAA 2544 Ser Ar~ Phe Gly Ser Ala Ala Ala Arg Ala Ser Ser Ser Leu Gly Gln 835 840 8~5 GGG TCT GGC TCC CAG TTT GTC CCC ACT GTT TGC AAT GGC CGT &AG GTG 2592 Gly Ser Gly Ser Gln Phe Val Pro Thr Val Cys Asn Gly Arg Glu Val Val Asp Ser Th- Th- Ser Ser Leu ~2) INFOP~ATION FO-K SEQ ID NO: 2:

~i) S~QUEN OE r~R~r~FRT~TICS:
(A) LENGTH: 872 amino acids (B) TYPE: amino ar-id (D) TOPOLOGY: lirear (ii) ~OLECULE TYPE: pro~ein (xi) SEQUENCE V~K1~L1UN: SEQ ID NO: 2:

Me~ Gly Ser Leu Leu Ala Leu Leu Ala Leu Leu Pro Leu Trp Gly Ala Val Ala Glu Gly Pro Ala Lys Lys Val Leu Thr L~eu Glu Gly Asp Leu q5 30 Val Leu Gly Gly Leu Phr2 P-o val His &lr Lys Gly Gly Pro Ala &lu ~ 45 WO96/06167 2 1 9 6 9 9 7 r~ 5~ &

Asp Cys Gly Pro Val Asn Glu His Arg Gly Ile Gln Arg Leu Glu Ala Met Leu Phe Ala Leu Asp Arg Ile Asn Arg Asp Pro His Leu Leu Pro ~ly Val Arg Leu Gly Ala His Ile Leu Asp Ser Cys Ser Lys Asp Thr g5 ~is Ala Leu Glu Gln Ala Leu Asp Phe Val Arg Ala Ser Leu Ser Arg Gly Ala Asp Gly Ser Arg His Ile Cys Pro Asp Gly Ser Tyr Ala Thr His Gly Asp Ala Pro Thr Ala Ile Thr Gly Val Ile Gly Gly Ser Tyr 130 135 ~ 140 Ser Asp Val Ser Ile Gln Val Ala Asn Leu Leu Arg Leu Phe Gln Ile ~ro Gln Ile Ser Tyr Ala Ser Thr Ser Ala Lys Leu Ser Asp Lys Ser ~rg Tyr Asp Tyr Phe Ala Arg Thr Val Pro Pro Asp Phe Phe Gln Ala Lys Ala Met Ala Glu Ile Leu Arg Phe Phe Asn Trp Thr Tyr Val Ser Thr Glu Ala Ser Glu Gly Asp Tyr Gly Glu Thr Gly Ile Glu Ala Phe Glu Leu Glu Ala Ar~ Ala Arg Asn Ile Cys Val Ala Thr Ser Glu Lys ... . _ . .. . . _ _ ... _: _ . . _ _ _ _, _ _ _ _ ~ WO96/06167 2 1 969 9-7~

Val Gly Arg Ala Met Ser Arg Ala Ala Phe Glu Gly Val Val Arg Ala Leu Leu Gln Lys Pro Sër Ala Arg Val Ala Val Leu Phe Thr Arg Ser Glu Asp Ala Arg Glu Leu Leu Ala Ala Ser Gln Arg Leu Asn Ala Ser phe Thr Trp Val Ala Ser Asp Gly Trp Gly Ala Leu Glu Ser Val Val Ala Gly Ser GlU Gly Ala Ala Glu Gly Ala Ile Thr Ile Glu Leu Ala Ser Tyr Pro Ile Ser Asp Phe Ala Ser Tyr Phe Glr. Ser Leu Asp Pro Trp Asn Asn Ser Arg Asn Pro Trp Phe Arg Glu Phe Trp Glu Gln Arg Phe Arg Cys Ser Phe Arg Gln Arg Asp Cys Ala Ala His Ser Leu Arg Ala Val Pro Phe Glu Gln Glu Ser Lys Ile Met Phe Val Val Asn Ala Val Tyr Ala Met Ala His Ala Leu His Asn Met ~is Arg Ala Leu Cys Pro Asn Thr Thr Arg Leu Cys Asp Ala Met Arg Pro val Asn Gly Arg 405 410 . 415 Arg Leu ~yr Lys Asp Phe Val Leu Asn Val Lys Phe Asp Ala Pro Phe 420 ~25 430 21 969~7 ' ~

Arg Pro Ala Asp Thr His Asr, Glu Val Arg Phe Asp Arg Phe Gly Asp 435 440 . 445 Gly Ile Gly Arg Tyr Asn Ile Phe Thr Tyr Leu Arg Ala Gly Ser Gly Arg Tyr Arg Tyr Gln Lys Val Gly Tyr Trp Ala Glu Gly Leu Thr Leu ~sp Thr Ser Leu Ile Pro Trp Ala Ser Pro Ser Ala Gly Pro Leu Ala ~la Ser Arg Cys Ser Glu Pro Cys Leu Gln Asn Glu Val Lys Ser Val Gln Pro Gly Glu Val Cys Cys Trp Leu Cys Ile Pro Cys Gln Pro Tyr Glu Tyr Arg Leu Asp Glu Phe Thr Cys Ala Asp Cys Gly Leu Gly Tyr Trp Pro Asn Ala Ser Leu Thr Gly Cys Phe Glu Leu Pro Gln Glu Tyr ~le Arg Trp Gly Asp Ala Trp Ala Val Gly Pro Val Thr Ile Ala Cys ~eu Gly Ala Leu Ala Thr Leu Phe Val Leu Gly Val Phe Val Arg His Asn Ala Thr Pro Val Val Lys Ala Ser Gly Arg Glu Leu Cys Tyr Ile 595 600 : 605 Leu Leu Gly Gly Val Phe Leu Cys Tyr Cys ~5et Thr Phe Ile Phe Ile 610 ~ 615 620 ~ WO 96106167 2 1 9 6 9 9 7 P .,~
r Ala Lys Pro Ser Thr Ala Val Cys Thr Leu Ars~ Arg Leu Gly Leu Gly 625 . - 630 635 640 Thr Ala Phe Ser Vai Cys Tyr Ser Ala Leu Leu Thr Lys Thr Asn Arg Ile Ala Arsr Ile Phe Gly Gly Ala Arg Glu Gly Ala Gln Ars~ Pro Arg Phe Ile Ser Pro Ala Ser Gln Val Ala Ile Cys Leu Ala Leu Ile Ser Gly Gln Leu Leu Ile Val Val Ala Trp Leu Val Val Glu Ala Pro Gly Thr Gly Lys Glu Thr Ala Pro Glu Arg Arg Glu Val Val Thr Leu Arg Cys Asn E~is Arg Asp Ala Ser Net Leu Gly Ser Leu Ala Tyr Asn Val Leu Leu Ile Ala Leu Cys Thr Leu Tyr Ala Phe Asn Thr Arg Lys Cys Pro Glu Asn Phe Asn Glu Ala Lys Phe Ile Gly Phe Thr ~et Tyr Thr Thr Cys Ile Ile Trp Leu Ala Leu Leu Pro Ile Phe Tyr Val Thr Ser Ser Asp Tyr Arg Val Gln Thr Thr Thr Met Cys Val Ser Val Ser Leu Ser Gly Ser Val Val Leu Gly Cys Leu Phe Ala Pro Lys Leu His Ile WO 96/06167 ',' ' i ~ P~
2 ! 96997 Ile Leu Phe Gln Pro GLn Lys Asn Val Val Ser His Arg Ala Pro Thr Ser ~rg Phe Gly Ser Ala Ala Ala Arg Ala Ser Ser Ser Leu Gly Gln Gly Ser Gly Ser Gln Phe Val Pro Thr Val Cy5 Asn Gly Arg Glu Val Val Asp Ser Thr Thr Ser Ser Leu

Claims (34)

Claims

1. Purified human metabotropic glutamate receptor (hmGluR) 2.

2. A receptor according to claim 1 which has the amino acid sequence set forth in SEQ ID
NO:2.

3. A variant of the receptor of claim 1 or claim 2.

4. Composition of matter comprising a receptor of claim 1.

5. Process for the preparation of a receptor of claim 1 comprising multiplication of a suitable host cell in vitro or in vivo.

6. Use of a receptor according to any of claims 1 to 3 for the screening for a compound which modulates the activity of said receptor.

7. A fusion protein comprising a receptor according to any of claims 1 to 3.

8. Nucleic acid comprising a nucleic acid coding for a receptor according to any of claims 1 to 3, or a fragment of said nucleic acid.

9. Nucleic acid according to claim 8, which is a DNA.

10. A DNA according to claim 9 having the nucleotide sequence set forth in SEQ ID NO: 1.

11. Nucleic acid probe comprising at least 14 contiguous bases of the DNA according to claim 9 or 10, or the complement thereof.

12. Process for the preparation of a nucleic acid according to claim 11.

13. A DNA according to claim 9 which is a hybrid vector.

14. A host cell comprising a DNA of claim 9.

15. A eukaryotic host cell according to claim 14 expressing a DNA coding for a protein according to claim 1.

16. A host cell transfected with a DNA of claim 9.

17. A host cell according to claim 16 which is a mammalian cell.

18. Use of a host cell according to claim 16 for the screening of a compound which modulates the activity of a receptor according to claim 1.

19. Process for the preparation of a host cell according to claim 14.

20. Purified mRNA complementary to the DNA according to claim 9.

21. A method for identifying DNA encoding the hmGluR subtype according to claim 1 comprising: contacting human DNA with a probe according to claim 11, and identifying DNA(s) which substantially hybridize to said probe.

22. A method for identifying compounds binding to hmGluR2 comprising use of the receptor protein according to claim 1 in a competitive binding assay.

23. An assay for identifying compounds which modulate the activity of the hmGluRaccording to claim 1 comprising - contacting the cells of claim 15 with at least one compound or signal whose ability to modulate the activity of said receptor is sought to be determined, and subsequently - analyzing cells for a difference in functional response mediated by said receptor.

24. Assay according to claim 23 comprising - contacting the cells of claim 15 with at least one compound or signal whose ability to modulate activity of hmGluR2 is sought to be determined, and subsequently - monitoring said cells for a change in the level of a particular second messenger.

25. A method for modulating the signal transduction activity of the hmGluR subtype according to claim 1 comprising contacting said subtype with an effective amount of at least one compound identified in the assay of claim 23.

26. An agonist, antagonist or allosteric modulator identified by the assay of claim 23.

27. A method for detecting a glutamate agonist or an allosteric modulator of hmGluR2 having agonistic activity comprising the steps of (a) exposing a compound to the hmGluR
of claim 1 coupled to a response pathway, under conditions and for a time sufficient to allow interaction of the compound with the receptor and an associated response through the pathway, and (b) detecting an increase or decrease in the stimulation of the response pathway resulting from the interaction of the compound with hmGluR2, relative to the asbsence of the tested compound and therefrom determining the presence of an agonist or an allosteric modulator.

28. A method for identifying a glutamate antagonist or an allosteric modulator of hmGluR2 having antagonistic activity, said method comprising the steps of (a) exposing a compound in the presence of a known glutamate agonist to the hmGluR according to claim 1 coupled to a response pathway, under conditions and for a time sufficient to allow interaction of the agonist with the receptor and an associated response through the pathway, and (b) detecting an inhibition of the stimulation of the response pathway by the agonist resulting from the interaction of the test compound with hmGluR2, relative to the stimulation of the response pathway induced by the glutamate agonist alone, and therefrom determining the presence of an antagonist or an allosteric modulator having antagonist-like activity.

29. An antibody directed against the protein of claim 1.

30. An antibody according to claim 29 which is a polyclonal antibody.

31. An antibody according to claim 29 which is a monoclonal antibody.

32. A method for modulating the signal transduction activity of the hmGluR subtype according to claim 1 comprising contacting said receptor with an antibody of claim 29.

33. A receptor according to claim 1 obtainable by recombinant DNA technology.

34. A transgenic non-human animal which does not express an endogenous mGluR2 gene but a nucleic acid encoding the receptor acording to any of claims 1 to 3.