Note: Descriptions are shown in the official language in which they were submitted.
WO 96/06167 ~ rl ~ ,o
~ I~ r. ~. ,_, ~
21 96997
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
- 8 -
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~
- 12-
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 ,.~ --
- 14-
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
- 19-
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
-20 -
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
21 96997
- 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~.~
- 23 -
~_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
-24-
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
-25 -
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
-28 -
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,; '
-29-
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 '~ - '
-30 -
~U~N~ LISTING
(1) GENERAL INFORNATION:
(i) APPLICANT: :
(A) NA~E: CIBA-GEIGY AG
(B) STREET: Klybeckstr. 141
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(G) TELEPHONE: +41 61 69 11 11
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(I) TELEX: 962 991
(ii) TITLE OF INVENTION: Glutamate Receptor
(iii) NUMBER OF ~U~N~:S: 2
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(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
-31-
(ix) FEATURE:
~A~ NAME/KEY: CDS
(B) LOCATION: l..26l8
(D) OTHER INFORMATION: /product= "hmGluR2
(Xi ) ~ ~N~ DESCRIPTION: SEQ ID NO: l:
ATG GGA TCG CTG CTT GCG CTC CTG GCA CTG CTG CCG CTG TGG GGT 5CT
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
- 70 75 80
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
-32-
CAT GCG CTG GAG CAG GCA CTG GAC TTT GTG CGT GCC TCA CTC AGC CGT 336
His Ala Leu Glu Gln Ala Leu Asp Phe Val Arg ~la Ser Leu Ser Arg
100 105 110
GGT GCT GAT GGA TCA CGC CAC ATC TGC CCC GAC GGC TCT TAT GCG ACC 384
Gly Ala Asp Gly Ser Arg His Ile Cys Pro Asp Gly Ser Tyr Ala Thr
115 120 125
CAT GGT GAT GCT CCC ACT GCC ATC ACT GGT GTT ATT GGC GGT TCC TAC 432
His Gly Asp Ala Pro Thr Ala Ile Thr Gly Val Ile Gly Gly Ser Tyr
130 135 140
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
145 150 155 160
CCA CAG ATT AGC TAC GCC TCT ACC AGT GCC AAG CTG AGT GAC AAG TCC 528
Pro Gln Ile Ser Tyr Ala Ser Thr Ser Ala Lys Leu Ser Asp Lys Ser
165 170 175
CGC TAT GAC TAC TTT GCC CGC ACA GTG CCT CCT GAC TTC TTC CAA GCC 576
Arg Tyr Asp Tyr Phe Ala Arg Thr Val Pro Pro Asp Phe Phe Gln Ala
180 185 190
AAG GCC ATG GCT GAG ATT CTC CGC TTC TTC AAC TGG ACC TAT GTG TCC 624
Lys Ala Met Ala Glu Ile Leu Arg Phe Phe Asn Trp Thr Tyr Val Ser
195 200 205
ACT GAG GCC TCT GAG GGC GAC TAT GGC GAG ACA GGC ATT GAG GCC TTT 672
Thr Glu Ala Ser Glu Gly Asp Tyr Gly Glu Thr Gly Ile Glu Ala Phe
210 215 220
GAG CTA GAG GCT CGT GCC CGC AAC ATC TGT GTG GCC ACC TCG GAG AAA 720
Glu Leu Glu Ala Arg Ala Arg Asn Ile Cys Val AIa Thr Ser Glu Lys
225 230 235 240
~ WO96/06167 2 ~ 96997 P~
-33-
GTG GGC CGT GCC ATG AGC CGC GCG GCC TTT GAG GGT GTG GTG CGA GCC 763
Val Gly Arg Ala Met Ser Arg Ala Ala Phe Glu Gly Val Val Arg Ala
245 250 255
CTG CTG CAG AAG CCC AGT GCC CGC GTG GCT GTC CTG TTC ACC CGT TCT 816
Leu Leu Gln Lys Pro Ser Ala Arg Val Ala Val Leu Phe Thr Ar~ Ser
260 265 270
GAG GAT GCC CGG GAG CTG CTT GCT GCC AGC CAG CGC CTC AAT GCC AGC 864
Glu AS~ Ala Arg Glu Leu Leu Ala Ala Ser Gln Arg Leu Asn Ala Ser
275 280 285
TTC ACC TGG GTG GCC AGT GAT GGT TGG GGG GCC CTG GAG AGT GTG GTG 912
Phe Thr Trp Val Ala Ser Asp Gly Trp Gly Ala Leu Glu Ser Val Val
290 295 300
GCA GGC AGT GAG GGG GCT GCT GAG GGT GCT ATC ACC ATC GAG CTG GCC 960
Ala Gly Ser Glu Gly Ala Ala Glu Gly Ala Ile Thr Ile Glu Leu Ala
305 . 310 315 320
TCC TAC CCC ATC AGT GAC TTT GCC TCC TAC TTC CAG AGC CTG GAC CCT 1008
Ser Tyr Pro Ile Ser Asp Phe Ala Ser Tyr Phe Gln Ser Leu Asp Pro
325 330 335
TGG AAC AAC AGC CGG AAC CCC TGG TTC CGT GAA TTC TGG GAG CAG AGG 1056
Trp Asn Asn Ser Arg Asn Pro Trp Phe Ar~ Glu Phe Trp Glu Gln Ar~
340 345 350
TTC CGC TGC AGC TTC CGG CAG CGA GAC TGC GCA GCC CAC TCT CTC CGG 1104
Phe Arg Cys Ser Phe Arg Gln Arg Asp Cys Ala Ala ~is Ser Leu Ar~
355 360 365
GCT GTG CCC TTT GAA CAG GAG TCC AAG ATC ATG TTT GTG GTC AAT GCA 1152
Ala Val Pro Phe Glu Gln Glu Ser Lys Ile ~et Phe Val Val Asn Ala
370 375 380
WO 96106167 2 1 9 6 9 9 7 . I P~~ ~"28
GTG TAC GCC ATG GCC CAT GCG CTC CAC AAC ATG CAC CGT GCC CTC TGC 1200
Val Tyr Ala Met Ala His Ala Leu His Asn Met His Arg Ala Leu Cys
385 390 395 400
CCC AAC ACC ACC CGG CTC TGT GAC GCG ATG CGG CCA GTT AAC GGG CGC 1248
Pro Asn Thr Thr Arg Leu Cys Asp Ala Met Arg Pro Val Asn Gly Arg
405 410 415
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
420 425 430
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
435 440 445
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
450 455 460
CGC TAT CGC TAC CAG AAG GTG GGC TAC TGG GCA GAA GGC TTG ACT CTG 1440
Arg Tyr Ar~ Tyr Gln Lys Val Gly Tyr Trp Ala Glu Gly Leu Thr Leu
465 470 475 480
GAC ACC AGC CTC ATC CCA TGG GCC TCA CCG TCA GCC GGC CCC CTG GCC 1488
Asp Thr Ser Leu Ile Pro Trp Ala Ser Pro Ser Ala Gly Pro Leu Ala
485 490 495
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
500 505 510
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
515 520 525
~ WO96/06167 2 1 969~7~ P~ 728
GAG TAC CGA TTG GAC GAA TTC ACT TGC GCT GAT TGT GGC CTG GGC TAC 1632
Glu Tyr Arg Leu ASp Glu Phe Thr Cys Ala ASp Cys Gly Leu Gly Tyr
530 535 , 540
TGG CCC AAT GCC AGC CTG ACT GGC TGC TTC GAA CTG CCC CAG GAG TAC 1680
Trp Pro Asn Ala Ser Leu Thr Gly Cys Phe Glu Leu Pro Gln Glu Tyr
545 550 555 560
ATC CGC TGG GGC GAT GCC TGG GCT GTG GGA CCT GTC ACC ATC GCC TGC 1728
Ile Arg Trp Gly Asp Ala Trp Ala Val Gly Pro Val Thr Ile Ala Cy8
565 570 575
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
580 585 590
AAT GCC ACA CCA GTG GTC AAG GCC TCA GGT CGG GAG CTC TGC TAC ATC 1824
Asn Ala Thr Pro Val Val Lys Ala Ser Gly Ar~ Glu Leu Cys Tyr Ile
595 600 605
CTG CTG GGT GGT GTC TTC CTC TGC TAC TGC ATG ACC TTC ATC TTC ATT 1872
Leu Leu Gly Gly Val Phe Leu Cys Tyr Cys Met Thr Phe Ile Phe Ile
610 615 620
GCC AAG CCA TCC ACG GCA GTG TGT ACC TTA CGG CGT CTT GGT TTG GGC 1920
Ala Lys Pro Ser Thr Ala Val Cys Thr Leu Arg Arg Leu Gly Leu Gly
625 630 635 640
ACT GCC TTC TCT GTC TGC TAC TCA GCC CTG CTC ACC AAG ACC AAC CGC 1968
Thr Ala Phe Ser Val Cys Tyr Ser Ala Leu Leu Thr Lys Thr Asn Arç~
6~5 650 655
.=
ATT GCA CGC ATC TTC GGT GGG GCC CGG GAG GGT GCC CAG CGG CCA CGC 2016
Ile Ala Arg Ile Phe Gly Gly Ala Arg Glu Gly Ala Gln ArsJ Pro Arg
660 665 670
WO96/06167 2 ! 9 6 9 9 7' - ' ~ ' ~ r ~
-36- _
TTC ATC AGT CCT GCC TCA CAG GTG GCC ATC TGC CTG GCA CTT ATC TCG 2064
Phe Ile Ser Pro Ala Ser Gln Val Ala Ile Cys Leu Ala Leu Ile Ser
675 680 685 -
GGC CAG CTG CTC ATC GTG GTC GCC TGG CTG GTG GTG GAG GCA CCG GGC 2112
Gly Gln Leu Leu Ile Val Val Ala Trp Leu Val Val Glu Ala Pro Gly
690 695 700
ACA GGC AAG GAG ACA GCC CCC GAA CGG CGG GAG GTG GTG ACA CTG CGC 2160
Thr Gly Lys Glu Thr Ala Pro Glu Arg Arg Glu Val Val Thr Leu Arg
705 710 715 720
TGC AAC CAC CGC GAT GCA AGT ATG TTG GGC TCG CTG GCC TAC AAT GTG 2208
Cys Asn HiS Arg Asp Ala Ser Met Leu Gly Ser Leu Ala Tyr Asn Val
725 730 735
CTC CTC ATC GCG CTC TGC ACG CTT TAT GCC TTC AAT ACT CGC AAG TGC 2256
Leu Leu Ile Ala Leu Cys Thr Leu Tyr Ala Phe Asn Thr Arg Lys Cys
740 745 750
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
755 760 765
ACC TGC ATC ATC TGG CTG GCA TTG TTG CCC ATC TTC TAT GTC ACC TCC 2352
Thr Cys Ile Ile Trp Leu Ala Leu Leu Pro Ile Phe Tyr Val Thr Ser
770 775 780
AGT GAC TAC CGG GTA CAG ACC ACC ACC ATG TGC GTG TCA GTC AGC CTC 2400
Ser Asp Tyr Arg Val Gl~ Thr Thr Thr Met CYs Val Ser Val Ser Leu
785 790 795 800
AGC GGC TCC GTG GTG CTT GGC TGC CTC TTT GCG CCC AAG CTG CAC ATC 2448
Ser Gly Ser Val Val Leu Gly Cys Leu Phe Ala Pro Lys Leu His Ile
805 810 815
21 9~997
W O96/06167 P~ 728
-37-
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
820 825 830
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
850 855 860
GTG GAC TCG ACA ACG TCA TCG CTT TG 2619
Val Asp Ser Th- Th- Ser Ser Leu
865 870
~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~ &
-38-
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
100 105 110
Gly Ala Asp Gly Ser Arg His Ile Cys Pro Asp Gly Ser Tyr Ala Thr
115 120 125
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
145 150 155 160
~ro Gln Ile Ser Tyr Ala Ser Thr Ser Ala Lys Leu Ser Asp Lys Ser
165 170 175
~rg Tyr Asp Tyr Phe Ala Arg Thr Val Pro Pro Asp Phe Phe Gln Ala
180 185 190
Lys Ala Met Ala Glu Ile Leu Arg Phe Phe Asn Trp Thr Tyr Val Ser
195 200 205
Thr Glu Ala Ser Glu Gly Asp Tyr Gly Glu Thr Gly Ile Glu Ala Phe
210 215 220
Glu Leu Glu Ala Ar~ Ala Arg Asn Ile Cys Val Ala Thr Ser Glu Lys
225 230 235 240
... . _ . .. . . _ _ ... _: _ . . _ _ _ _, _ _ _ _
~ WO96/06167 2 1 969 9-7~
-39 -
Val Gly Arg Ala Met Ser Arg Ala Ala Phe Glu Gly Val Val Arg Ala
245 250 255
Leu Leu Gln Lys Pro Sër Ala Arg Val Ala Val Leu Phe Thr Arg Ser
260 265 270
Glu Asp Ala Arg Glu Leu Leu Ala Ala Ser Gln Arg Leu Asn Ala Ser
275 280 285
phe Thr Trp Val Ala Ser Asp Gly Trp Gly Ala Leu Glu Ser Val Val
290 295 300
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 Glr. Ser Leu Asp Pro
325 330 335
Trp Asn Asn Ser Arg Asn Pro Trp Phe Arg Glu Phe Trp Glu Gln Arg
340 345 350
Phe Arg Cys Ser Phe Arg Gln Arg Asp Cys Ala Ala His Ser Leu Arg
355 360 365
Ala Val Pro Phe Glu Gln Glu Ser Lys Ile Met Phe Val Val Asn Ala
370 375 380
Val Tyr Ala Met Ala His Ala Leu His Asn Met ~is Arg Ala Leu Cys
385 390 395 - 400
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
WO 96/06167
21 969~7 ' ~
-40 -
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
450 455 460
Arg Tyr Arg Tyr Gln Lys Val Gly Tyr Trp Ala Glu Gly Leu Thr Leu
465 470 475 480
~sp Thr Ser Leu Ile Pro Trp Ala Ser Pro Ser Ala Gly Pro Leu Ala
485 490 495
~la Ser Arg Cys Ser Glu Pro Cys Leu Gln Asn Glu Val Lys Ser Val
500 505 510
Gln Pro Gly Glu Val Cys Cys Trp Leu Cys Ile Pro Cys Gln Pro Tyr
515 520 525
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
545 550 555 560
~le Arg Trp Gly Asp Ala Trp Ala Val Gly Pro Val Thr Ile Ala Cys
565 570 575
~eu Gly Ala Leu Ala Thr Leu Phe Val Leu Gly Val Phe Val Arg His
580 585 590
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
-41 -
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
645 650 655
Ile Ala Arsr Ile Phe Gly Gly Ala Arg Glu Gly Ala Gln Ars~ Pro Arg
660 665 670
Phe Ile Ser Pro Ala Ser Gln Val Ala Ile Cys Leu Ala Leu Ile Ser
675 680 685
Gly Gln Leu Leu Ile Val Val Ala Trp Leu Val Val Glu Ala Pro Gly
690 695 700
Thr Gly Lys Glu Thr Ala Pro Glu Arg Arg Glu Val Val Thr Leu Arg
705 710 715 720
Cys Asn E~is Arg Asp Ala Ser Net Leu Gly Ser Leu Ala Tyr Asn Val
725 730 735
Leu Leu Ile Ala Leu Cys Thr Leu Tyr Ala Phe Asn Thr Arg Lys Cys
740 745 750
Pro Glu Asn Phe Asn Glu Ala Lys Phe Ile Gly Phe Thr ~et Tyr Thr
755 760 765
Thr Cys Ile Ile Trp Leu Ala Leu Leu Pro Ile Phe Tyr Val Thr Ser
770 775 780
Ser Asp Tyr Arg Val Gln Thr Thr Thr Met Cys Val Ser Val Ser Leu
785 790 795 800
Ser Gly Ser Val Val Leu Gly Cys Leu Phe Ala Pro Lys Leu His Ile
805 810 815
WO 96/06167 ',' ' i ~ P~
2 ! 96997
-42-
Ile Leu Phe Gln Pro GLn Lys Asn Val Val Ser His Arg Ala Pro Thr
820 825 830
Ser ~rg Phe Gly Ser Ala Ala Ala Arg Ala Ser Ser Ser Leu Gly Gln
835 840 845
Gly Ser Gly Ser Gln Phe Val Pro Thr Val Cy5 Asn Gly Arg Glu Val
850 855 860
Val Asp Ser Thr Thr Ser Ser Leu
865 870
