Title: Human metabotropic glutamate receptor 7 subtypes
United States Patent: 6,515,107
Issued: February 4, 2003
Inventors: Flor; Peter Josef (Freiburg, DE); Kuhn; Rainer (Lorrach,
DE); Lindauer; Kristin (Basel, CH); Puttner; Irene (Basel, CH); Knopfel;
Thomas (Rheinfelden, CH)
Assignee: Novartis Corporation (New York, NY)
Appl. No.: 817464
Filed: March 26, 2001
The present invention relates to human metabotropic glutamate receptor (hmGluR)
proteins, isolated nucleic acids coding therefor, host cells producing the
proteins of the invention, methods for the preparation of such proteins,
nucleic acids and host cells, and uses thereof.
Description of the Invention
The present invention relates to human metabotropic glutamate receptor (hmGluR)
proteins, isolated nucleic acids coding therefor, host cells producing the
proteins of the invention, methods for the preparation of such proteins,
nucleic acids and host cells, and uses thereof. Furthermore, the invention
provides antibodies directed against the hmGluR proteins of the invention.
Metabotropic glutamate receptors (hmGluR) belong to the class of G-protein
(guanine nucleotide binding protein) coupled receptors which upon binding of
a glutamatergic ligand may transduce an extracellular signal via an
intracellular second messenger system such as calcium ions, a cyclic
nucleotide, diacylglycerol and inositol 1,4,5-triphosphate into a
physiological response. Possessing seven putative transmembrane spanning
segments, preceded by a large extracellular amino-terminal domain and
followed by a large carboxy-terminal domain metabotropic glutamate receptors
are characterized by a common structure. Based on the degree of sequence
identity at the amino acid level the class of mGluR can be divided into
different subfamilies comprising individual receptor subtypes (Nakanishi,
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 amino acid sequences are less than
about 50% identical. Within a subfamily the degree of sequence identity is
generally less than about 70%. Thus a particular subtype may be
characterized by its amino acid sequence homology to another mGluR subtype,
especially a subtype of the same mammalian species. Furthermore, a
particular subtype may be characterized by its region and tissue
distribution, its cellular and subcellular expression pattern or by its
distinct physiological profile, e.g. by its electrophysiological and
The amino acid L-glutamate being the major excitatory neurotransmitter,
glutamatergic systems are presumed to play an important role in numerous
neuronal processes including fast excitatory synaptic transmission,
regulation of neurotransmitter releases, long-term potentation, learning and
memory, developmental synaptic plasticity, hypoxic-ischemic damage and
neuronal cell death, epileptiform seizures, as well as the pathogenesis of
several neurodegenerative disorders. Up to today, no information is
available on human metabotropic glutamate receptor (hmGluR) subtypes, e.g.
on their amino acid sequence or tissue distribution. This lack of knowledge
particularly hampers the search for human therapeutic agents capable of
specifically influencing any disorder attributable to a defect in the
glutamatergic system. In view of the potential physiological and
pathological significance of metabotropic glutamate receptors, there is a
need for human receptor subtypes and cells producing such subtypes in
amounts sufficient for elucidating the electrophysiological and
pharmacological properties of these proteins. For example, drug screening
assays require purified human receptor proteins in an active form, which
have not yet been attainable.
It is an object of the present invention to fulfill this need, namely to
provide distinct hmGluR subtypes, nucleic acids coding therefor and host
cells producing such subtypes. In particular, the present invention
discloses the hmGluR subfamily comprising the subtype designated hmGluR4,
and the individual proteins of said subfamily. In the following, said
subfamily will be referred to as the hmGluR4 subfamily. Contrary to other
hmGluR subtypes the members of this subfamily are potently activated by
L-2-amino-4-phosphobutyric acid (AP4) and, when expressed 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 comprising a
recombinant hmGluR subtype of the invention in screening for hmGluR reactive
drugs offers (among others) the possibilities 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
(potentially resulting in greater biological and disease specificity).
More specifically, the present invention relates to a hmGluR subtype
characterized in that its amino acid sequence is more than about 65%
identical to the sequence of the hmGluR4 subtype having the amino acid
sequence depicted in SEQ ID NO:2.
According to the invention 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 glutamatergic ligand transduces an extracellular
signal via an intracellular second messenger system. In such case, a subtype
of the invention is characterized in that it modifies the level of a cyclic
nucleotide (cAMP, cGMP). Alternatively, signal transduction may occur via
direct interaction of the G protein coupled to a receptor subtype of the
invention with another membrane protein, such as an ion channel or another
receptor. A receptor subtype of the invention is believed to be encoded by a
distinct gene which does not encode another metabotropic glutamate receptor
subtype. A particular subtype of the invention may be characterized by its
distinct physiological profile, preferably by its signal transduction and
pharmacological properties. Pharmacological properties are e.g. the
selectivity for agonists and antagonist responses.
As defined herein, a glutamatergic ligand is e.g. L-glutamate or another
compound interacting with, and particularly binding to, a hmGluR subtype in
a glutamate like manner, such as ACPD
(1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid), an ACPD-like ligand, e.g.
QUIS (quisqualate), AP4, and the like. Other ligands, e.g. (R,S)-.alpha.-methylcarboxyphenylglycine
(MCPG) or .alpha.-methyl-L-AP4, may interact with a receptor of the
invention in such a way that binding of glutamatergic ligand is prevented.
As used hereinbefore or hereinafter, 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 engineering. The
purified proteins, DNAs and RNAs of the invention may be useful in ways that
the proteins, DNAs and RNAs as they naturally occur are not, such as
identification of compounds selectively modulating the expression or the
activity of a hmGluR of the invention.
Purified hmGluR of the invention means a member of the hmGluR4 subfamily
which has been identified and is free of one or more components of its
natural environment Purified hmGluR includes purified hmGluR of the
invention in recombinant cell culture. The enriched form of a subtype of the
invention refers to a preparation containing said subtype in a concentration
higher than natural, e.g. a cellular membrane fraction comprising said
subtype. If said subtype is in a pure form it is substantially free from
other macromolecules, particularly from naturally occurring proteinaceous
contaminations. If desired, the subtype of the invention may be solubilized.
A preferred purified hmGluR subtype of the invention is a recombinant
protein. Preferably, the subtype of the invention is in an active state
meaning that it has both ligand binding and signal transduction 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
Preferred hmGluR subtypes of the hmGluR4 subfamily are subtypes hmGluR4,
hmGluR7 and hmGluR6. A particularly preferred hmGluR4 subtype is the protein
having the amino acid sequence set forth in SEQ ID NO:2. A hmGluR7-type
protein may comprise a polypeptide selected from the group consisting of the
polypeptides having the amino acid sequences depicted in SEQ ID NOs: 4, 6, 8
and 10, respectively. Such hmGluR7 subtype is preferred. Particularly
preferred are the hmGluR7 subtypes having the amino acid sequences set forth
in SEQ ID NOs: 12 and 14, respectively. A preferred hmGluR6-type protein
comprises a polypeptide having the amino acid sequence depicted in SEQ ID
The invention is further intended to include variants of the receptor
subtypes of the invention. For example, a variant of a hmGluR subtype of the
invention is a functional or immunological equivalent of said subtype. A
functional equivalent is a protein, particularly a human protein, displaying
a physiological profile essentially identical to the profile characteristic
of said particular subtype. The physiological profile in vitro and in vivo
includes receptor effector function, electrophysiological and
pharmacological properties, e.g. selective interaction with agonists or
antagonists. Exemplary functional equivalents may be splice variants encoded
by mRNA generated by alternative splicing of a primary transcript, amino
acid mutants and glycosylation variants. An immunological equivalent of a
particular hmGluR subtype is a protein or peptide capable of generating
antibodies specific for said subtype. Portions of the extracellular domain
of the receptor, e.g. peptides consisting of at least 6 to 8 amino acids,
particularly 20 amino acids, are considered particularly useful
Further variants included herein are membrane-bound and soluble fragments
and covalent or aggregative conjugates with other chemical moieties, these
variants displaying one or more receptor functions, such as ligand binding
or signal transduction. Exemplary fragments of hmGluR subtypes of the
invention are the polypeptides having the amino acid sequences set forth in
SEQ ID NOs: 4, 6, 8, 10 and 16, respectively. The fragments of the invention
are obtainable from a natural source, by chemical synthesis or by
recombinant techniques. Due to their capability of competing with the
endogenous counterpart of a hmGluR subtype of the invention for its
endogenous ligand, fragments, or derivatives thereof, comprising the ligand
binding domain are envisaged as therapeutic agents.
Covalent derivatives include for example aliphatic esters or amides of a
receptor carboxyl group, O-acyl derivatives of hydroxyl group containing
residues and N-acyl derivatives of amino group containing residues. Such
derivatives can be prepared by linkage of functionalities to reactable
groups which are found in the side chains and at the N- and C-terminus of
the receptor protein. The protein of the invention can also be labeled with
a detectable group, for example radiolabeled, covalently bound to rare earth
chelates or conjugated to a fluorescent moiety.
Further derivatives are covalent conjugates of a protein of the invention
with another protein or peptide (fusion proteins). Examples are fusion
proteins comprising different portions of different glutamate receptors.
Such fusion proteins may be useful for changing the coupling to G-proteins
and/or improving the sensitivity of a functional assay. For example, in such
fusion proteins or chimeric receptors, the intracellular domains of a
subtype of the invention may be replaced with the corresponding domains of
another mGluR subtype, particularly another hmGluR subtype, e.g. a hmGLuR
subtype belonging to another subfamily. Particularly suitable for the
construction of such a chimeric receptor are the intracellular domains of a
receptor which activates the phospholipase C/Ca2+ signaling
pathway, e.g. mGluR1 (Masu et al., Nature 349,760-765) or mGluR5. An
intracellular domain suitable for such an exchange is e.g. the second
intracellular loop, also referred to as i2 (Pin et al., EMBO J. 13, 342-348
(1994)). Thus it is possible to analyze the interaction of a test 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
synthesized by recombinant techniques or agents known in the art as being
suitable for crosslinking proteins.
Aggregative derivatives are e.g. adsorption complexes with cell membranes.
In another embodiment, the present invention relates to a composition of
matter comprising a hmGluR subtype of the invention.
The proteins of the invention are useful e.g. as immunogens, in drug
screening assays, as reagents for immunoassays and in purification methods,
such as for affinity purification of a binding ligand.
A protein of the invention is obtainable from a natural source, e.g. by
isolation from brain tissue, by chemical synthesis or by recombinant
The invention further provides a method for preparing a hmGluR subtype of
the invention characterized in that suitable host cells producing a receptor
subtype of the invention are multiplied in vitro or in vivo. Preferably, the
host cells are transformed (transfected) with a hybrid vector comprising an
expression cassette comprising a promoter and a DNA sequence coding for said
subtype which DNA is controlled by said promoter. Subsequently, the hmGluR
subtype of the invention may be recovered. Recovery comprises e.g. isolating
the subtype of the invention from the host cells or isolating the host cells
comprising the subtype, e.g. from the culture broth. Particularly preferred
is a method for preparation of a functionally active receptor.
HmGluR muteins may be produced from a DNA encoding a hmGluR protein of the
invention which DNA has been subjected to in vitro mutagenesis resulting
e.g. in an addition, exchange and/or deletion of one or more amino acids.
For example, substitutional, deletional and insertional variants of a hmGluR
subtype of the invention are prepared by recombinant methods and screened
for immuno-crossreactivity with the native forms of the hmGluR.
A protein of the invention may also be derivatized in vitro according to
conventional methods known in the ar
Suitable host cells include eukaryotic cells, e.g. animal cells, plant cells
and fungi, and prokaryotic cells, such as gram-positive and gram-negative
bacteria, e.g. E. coli. Preferred eukaryotic host cells are of amphibian or
As used herein, in vitro means ex vivo, thus including e.g. cell culture and
tissue culture conditions.
This invention further covers a nucleic acid (DNA, RNA) comprising a
purified, preferably recombinant, nucleic acid (DNA, RNA) coding for a
subtype of the invention, or a fragment of such a nucleic acid. In addition
to being useful for the production of the above recombinant hmGluR proteins,
these nucleic acid are useful as probes, thus readily enabling those skilled
in the art to identify and/or isolate nucleic acid encoding a hmGluR protein
of the invention. The nucleic acid may be unlabeled or labeled with a
detectable moiety. Furthermore, nucleic acid according to the invention is
useful e.g. in a method for determining the presence of hmGluR, said method
comprising hybridizing the DNA (or RNA) encoding (or complementary to)
hmGluR to test sample nucleic acid and to determine the presence of hmGluR.
Purified hmGluR encoding nucleic acid of the invention includes nucleic acid
that is free from at least one contaminant nucleic acid with which it is
ordinarily associated in the natural source of hmGluR nucleic acid. Purified
nucleic acids thus is present in other than in the form or setting in which
it is found in nature. However, purified hmGluR nucleic acid embraces hmGluR
nucleic acid in ordinarily hmGluR expressing cells where the nucleic acid is
in a chromosomal location different from that of natural cells or is
otherwise flanked by a different DNA sequence than that found in nature.
In particular, the invention provides a purified or isolated DNA molecule
encoding a hmGluR subtype 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 complementary DNA thereto, or this
complementary (single stranded) DNA itself. Preferred is a DNA coding for
the above captioned preferred hmGluR subtypes, or a fragment thereof.
Furthermore, the invention relates to a DNA comprising such a DNA.
More specifically, preferred is a DNA coding for a hmGluR4 subtype or a
portion thereof, particularly a DNA encoding the hmGluR4 subtype having the
amino acid sequence set forth in SEQ ID NO:2, e.g. the DNA with the
nucleotide sequence set forth in SEQ ID NO:1. An exemplary DNA fragment
coding for a portion of hmGluR4 is the hmGluR4-encoding portion of cDNA
cmR20 as described in the Examples.
Equally preferred is a DNA encoding a hmGluR7 subtype, particularly a DNA
encoding any of the hmGluR7 subtypes having the amino acid sequences set
forth in SEQ ID NOs: 12 and 14, respectively, e.g. the DNAs with the
nucleotide sequences set forth in SEQ ID NOs: 11 and 13, respectively. The
invention further provides a DNA fragment encoding a portion of a hmGluR7
subtype, particularly the hmGluR7 subtypes identified as preferred above.
Exemplary hmGluR7 DNA fragments include the hmGluR7-encoding portions of
cDNAs cmR2, cmR3, cmR5 and cR7PCR1, as described in the Examples, or a DNA
fragment which encodes substantially the same amino acid sequence as that
encoded by the hmGluR7-encoding portion of plasmid cmR2 deposited with the
DSM on Sep. 13, 1993, under accession number DSM 8550. These DNAs encode
portions of putative splice variants of the hmGluR7 subtype described
Also preferred is a DNA encoding a hmGluR6 subtype or a portion thereof,
particularly a DNA encoding the portion of the hmGluR6 subtype, the amino
acid sequence of which is depicted in SEQ ID NO:16, or a DNA which encodes
substantially the same amino acid sequence as that encoded by the
hmGluR6-encoding portion of plasmid cmR1 deposited with the DSM on Sep. 13,
1993, under accession number DSM 8549. An exemplary DNA sequence is set
forth in SEQ ID NO:15.
The nucleic acid sequences provided herein may be employed to identify DNAs
encoding further hmGluR subtypes. For example, nucleic acid sequences of the
invention may be used for identifying DNAs encoding further hmGluR subtypes
belonging to the subfamily comprising hmGluR 4. A method for identifying
such DNA comprises contacting human DNA with a nucleic acid probe described
above and identifying DNA(s) which hybridize to that probe.
Exemplary nucleic acids of the invention can alternatively be characterized
as those nucleic acids which encode a hmGluR subtype of the invention and
hybridize to a DNA sequence set forth in SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13
or 15, or a selected portion (fragment) of said DNA sequence. For example,
selected fragments useful for hybridization are the protein-encoding
portions of said DNAs. Preferred are such DNAs encoding a hmGluR of the
invention which hybridize under high-stringency conditions to the
Stringency of hybridization refers to conditions under which polynucleic
acids hybrids are stable. Such conditions are evident to those of ordinary
skill in the field. As known to those of skill in the art, the stability of
hybrids is reflected in the melting temperature (Tm) of the hybrid
which decreases approximately 1 to 1.5oC. with every 1% decrease in
sequence homology. In general, the stability of a hybrid is a function of
sodium ion concentration and temperature. Typically, the hybridization
reaction is performed under conditions of higher stringency, followed by
washes of varying stringency.
As used herein, high stringency refers to conditions that permit
hybridization of only those nucleic acid sequences that form stable hybrids
in 1 M Na+ at 65-68oC. High stringency conditions can be
provided, for example, by hybridization in an aqueous solution containing
6.times.SSC, 5.times.Denhardt's, 1% SDS (sodium dodecyl sulfate), 0.1 Na+
pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific
competitor. Following hybridization, high stringency washing may be done in
several steps, with a final wash (about 30 min) at the hybridization
temperature in 0.2-0.1.times.SSC, 0.1% SDS.
Moderate stringency refers to conditions equivalent to hybridization in the
above described solution but at about 60-62oC. In that case the
final wash is performed at the hybridization temperature in 1.times.SSC,
Low stringency refers to conditions equivalent to hybridization in the above
described solution at about 50-52oC. In that case, the final wash is
performed at the hybridization temperature in 2.times.SSC, 0.1% SDS.
It is understood that these conditions may be adapted and duplicated using a
variety of buffers, e.g. formamide-based buffers, and temperatures.
Denhart's solution and SSC are well known to those of skill in the art as
are other suitable hybridization buffers (see, e.g. Sambrook, J., Fritsch,
E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd
edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA, or
Ausubel, F. M., et al. (1993) Current Protocols in Molecular Biology, Greene
and Wiley, USA). Optimal hybridization conditions have to be determined
empirically, as the length and the GC content of the probe also play a role.
Given the guidance 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 preparation of such
For example, a DNA of the invention is obtainable by chemical synthesis, by
recombinant DNA technology or by polymerase chain reaction (PCR).
Preparation by recombinant DNA technology may involve screening a suitable
cDNA or genomic library. A suitable method for preparing a DNA or of the
invention comprises the synthesis of a number of oligonucleotides, their
amplification by PCR methods, and their splicing to give the desired DNA
sequence. Suitable libraries are commercially available, e.g. the libraries
employed in the Examples, or can be prepared from neural or neuronal tissue
samples, e.g. hippocampus and cerebellum tissue, cell lines and the like.
For individual hmGluR subtypes (and splice variants) of the invention 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
advantageous to screen libraries prepared from different suitable tissues or
cells. As a screening probe, there may be employed a DNA or RNA comprising
substantially the entire coding region of a hmGluR subtype of the invention,
or a suitable oligonucleotide probe based on said DNA. A suitable
oligonucleotide probe (for screening involving hybridization) is a single
stranded DNA or RNA that has a sequence of nucleotides that includes at
least 14 contiguous bases that are the same as (or complementary to) any 14
or more contiguous bases set forth in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11,
13 and 15. The probe may be labeled with a suitable chemical moiety for
ready detection. The nucleic acid sequences selected as probes should be of
sufficient length and sufficiently unambiguous so that false positive
results are minimized.
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 clones 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
hybridization. For example, a suitable label means is a radiolabel. The
preferred method of labelling a DNA fragment is by incorporating 32 P-labelled
.alpha.-dATP with the Klenow fragment of DNA polymerase in a random priming
reaction, as is well known in the art. Oligonucleotides are usually
end-labeled with 32 P -labeled .gamma.-ATP and polynucleotide kinase.
However, other methods (e.g. non-radioactive) may also be used to label the
fragment or oligonucleotide, including e.g. enzyme labelling and
After screening the library, e.g. with a portion of DNA including
substantially the entire hmGluR-encoding sequence or a suitable
oligonucleotide based on a portion of said DNA, positive clones are
identified by detecting a hybridization signal; the identified clones are
characterized by restriction enzyme mapping and/or DNA sequence analysis,
and then examined, e.g. by comparison with the sequences set forth herein,
to ascertain whether they include DNA encoding a complete hmGluR (i.e., if
they include translation initiation and termination codons). If the selected
clones are incomplete, they may be used to rescreen the same or a different
library to obtain overlapping clones. If the library is genomic, then the
overlapping clones may include exons and introns. If the library is a cDNA
library, then the overlapping clones will include an open reading frame. In
both instances, complete clones may be identified by comparison with the
DNAs and deduced amino acid sequences provided herein.
Furthermore, in order to detect any abnormality of an endogenous hmGluR
subtype of the invention genetic screening may be carried out using the
nucleotide sequences of the invention as hybridization probes. Also, based
on the nucleic acid sequences provided herein antisense-type therapeutic
agents may be designed.
It is envisaged that the nucleic acid of the invention can be readily
modified by nucleotide substitution, nucleotide deletion, nucleotide
insertion or inversion of a nucleotide stretch, and any combination thereof.
Such modified sequences can be used to produce a mutant hmGluR subtype which
differs from the receptor subtypes found in nature. Mutagenesis may be
predetermined (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 complementary regions that could hybridize to produce secondary
mRNA structures such as loops or hairpins.
The cDNA or genomic DNA encoding native or mutant hmGluR of the invention
can be incorporated into vectors for further manipulation. Furthermore, the
invention concerns a recombinant DNA which is a hybrid vector comprising at
least one of the above mentioned DNAs.
The hybrid vectors of the invention comprise an origin of replication or an
autonomously replicating sequence, one or more dominant marker sequences
and, optionally, expression control sequences, signal sequences and
additional restriction sites.
Preferably, the hybrid vector of the invention comprises an above described
nucleic acid insert operably linked to an expression control sequence, in
particular those described hereinafter.
Vectors typically perform two functions in collaboration with compatible
host cells. One function is to facilitate the cloning of the nucleic acid
that encodes the hmGluR subtype of the invention, i.e. to produce usable
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 maintenance as an extrachromosomal element or by integration
into the host chromosome (expression vectors). A cloning vector comprises
the DNAs as described above, an origin of replication or an autonomously
replicating sequence, selectable marker sequences, and optionally, signal
sequences and additional restriction sites. An expression vector
additionally comprises expression control sequences essential for the
transcription and translation of the DNA of the invention. Thus an
expression vector refers to a recombinant DNA construct, such as a plasmid,
a phage, recombinant virus or other vector that, upon introduction into a
suitable host cell, results in expression of the cloned DNA. Suitable
expression vectors are well known in the art and include those that are
replicable in eukaryotic and/or prokaryotic cells.
Most expression vectors are capable of replication in at least one class of
organisms but can be transfected into another organism for expression. For
example, a vector is cloned in E. coli and then the same vector is
transfected into yeast or mammalian cells even though it is not capable of
replicating independently of the host cell chromosome. 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 exogenously
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 cells without any replication component
Advantageously, expression and cloning vector contain a selection gene also
referred to as selectable marker. This gene encodes a protein necessary for
the survival or growth of transformed host cells grown in a selective
culture medium. Host cells not transformed with the vector containing 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, methotrexate or tetracycline, complement
auxotrophic deficiencies, or supply critical nutrients not available from
Since the amplification of the vectors is conveniently done in E. coli. an
E. coli genetic marker and an E. coli origin of replication are
advantageously included. These can be obtained from E. coli plasmids, such
as pBR322, Bluescript vector or a pUC plasmid.
Suitable selectable markers for mammalian cells are those that enable the
identification of cells competent to take up hmGluR nucleic acid, such as
dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase,
or genes confering resistance to G418 or hygromycin. The mammalian cell
transfectants are placed under selection pressure which only those
tansfectants are uniquely adapted to survive which have taken up and are
expressing the marker.
Expression and cloning vectors usually contain a promoter that is recognized
by the host organism and is operably linked to hmGluR nucleic acid. Such
promoter may be inducible or constitutive. The promoters are operably linked
to DNA encoding hmGluR by removing the promoter from the source DNA by
restriction enzyme digestion and inserting the isolated promoter sequence
into the vector. Both the native hmGluR promoter sequence and many
heterologous promoters may be used to direct amplification and/or expression
of hmGluR DNA. However, heterologous promoters are preferred, because they
generally allow for greater transcription and higher yields of expressed
hmGluR as compared to native hmGluR promoter.
Promoters suitable for use with prokaryotic hosts include, for example, the
.beta.-lactamase and lactose promoter systems, alkaline phosphatase, 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 generally contain a Shine-Delgarno
sequence operably linked to the DNA encoding hmGluR.
HmGluR gene transcription from vectors in mammalian 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 a hmGluR subtype of the invention in eukaryotic host cells,
particularly mammalian cells, are e.g. cytomegalovirus (CMV)
promoter-containing vectors, RSV promoter-containg vectors and SV40
promoter-containing vectors and MMTV LTR promoter-containing vectors.
Depending on the nature of their regulation, promoters may be constitutive
or regulatable by experimental conditions.
Transcription of a DNA encoding a hmGluR subtype according to the invention
by higher eukaryotes may be increased by inserting an enhancer sequence into
The various DNA segments of the vector DNA are operatively linked, i.e. they
are contiguous and placed into a functional relationship to each other.
Construction of vectors according to the invention employs conventional
ligation techniques. Isolated plasmids or DNA fragments are cleaved,
tailored, and religated in the form desired to generate the plasmids
required. If desired, analysis to confirm correct sequences in the
constructed plasmids is performed in a manner known in the art. Suitable
methods for constructing expression vectors, preparing in vitro transcripts,
introducing DNA into host cells, and performing analyses for assessing
hmGluR expression and function are known to those skilled in the art. Gene
presence, amplification and/or expression may be measured in a sample
directly, for example, by conventional Southern blotting, northern blotting
to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis),
in situ hybridization, using an appropriately labelled probe based on a
sequence provided herein, binding assays, immunodetection and functional
assays. Suitable methods include those decribed in detail in the Examples.
Those skilled in the art will readily envisage how these methods may be
modified, if desired.
The invention further provides host cells capable of producing a hmGluR
subtype of the invention and including heterologous (foreign) DNA encoding
The nucleic acids of the invention can be expressed in a wide variety of
host cells, e.g. those mentioned above, that are transformed or transfected
with an appropriate expression vector. The receptor of the invention (or a
portion thereof) may also be expressed as a fusion protein. Recombinant
cells can then be cultured under conditions whereby the protein (s) encoded
by the DNA of the invention is (are) expressed.
Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-prositive
organisms, such as E. coli, e.g. E. coli, K-12 strains, DH5.alpha. and HB
101, or Bacilli. Further host cells suitable for hmGluR encoding vectors
include eukaryotic microbes such as filamentous fungi or yeast, e.g.
Saccharomyces cerevisiae. Higher eukaryotic cells include insect, amphebian
and vertebrate cells, particularly mammalian cells, e.g. neuroblastoma cell
lines or fibroblast derived cell lines. Examples of preferred mammalian cell
lines are e.g. HEK 293 cells, CHO cells, CV1 cells, BHK cells, L cells,
LLCPK-1 cells, GH3 cells, L cells and COS cells. In recent years propagation
of vertebrate cells in culture (tissue culture) has become a routine
procedure. The host cells referred to in this application comprise cells in
in vitro culture as well as cells that are within a host animal.
Suitable host cells for expression of an active recombinant hmGluR of the
invention advantageously express endogenous or recombinant G-proteins.
Preferred are cells producing little, if any, endogenous metabotropic
glutamate receptor. DNA may be stably incorporated into the cells or may be
transiently expressed according to conventional methods.
Stably transfected mammalian cells may be prepared by transfecting 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 transfectants, mammalian cells are transfected
with a reporter gene to monitor transfection 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 determined and optimized for a particular cell
A DNA of the invention may also be expressed in non-human transgenic
animals, particularly 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 appropriately positioned expression control
sequences, is introduced into pronuclei of fertilized eggs. Introduction may
be achieved, e.g. by microinjection. Integration of the injected DNA is
detected, e.g. by blot analysis of DNA from suitable tissue samples. It is
preferred that the introduced DNA be incorporated into the germ line of the
animal so that it is passed to the animal's progeny. Preferably, a
transgenic animal is developped by targeting a mutation to disrupt a hmGluR
sequence. Such an animal is useful e.g. for studying the role of a
metabotropic receptor in metabolism.
Furthermore, a knock-out animal may be developed by introducing a mutation
in the hmGluR sequence, thereby generating an animal which does not express
the functional hmGluR gene anymore. Such knock-out animal is useful e.g. for
studying the role of metabotropic receptor in metabolism. methods for
producing knock-out mice are known in the art.
Host cells are transfected or transformed with the above-captioned
expression or cloning vectors of this invention and cultured in conventional
nutrient media modified as appropriate for inducing promoters, selecting
transformants, 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 transfection with a vector encoding a heterologous DNA by
the calcium phosphate coprecipitation technique, by electroporation or by
lipofectin-mediated. Numerous methods of transfection are known to the
skilled worker in the field. Successful transfection is generally recognized
when any indication of the operation of this vector occurs in the host cell.
Transformation is achieved using standard techniques appropriate to the
particular host cells used.
Incorporation of cloned DNA into a suitable expression vector, transfection
of eukaryotic cells with a plasmid vector or a combination of plasmid
vectors, each encoding one or more distinct genes or with linear DNA, and
selection of transfected cells are well known in the art (see, e.g. Sambrook
et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor Laboratory Press).
Transfected or transformed cells are cultured using media and culturing
methods known in the art, preferably under conditions, whereby hmGluR
encoded by the DNA is expressed. The composition of suitable media is known
to those in the art, so that they can be readily prepared. Suitable
culturing media are also commercially 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, particularly mammalian
expression systems, including commercially available systems and other
systems known to those of skill in the art.
Human mGluR DNA of the invention is ligated into a vector, and introduced
into suitable host cells to produce transformed cell lines that express a
particular hmGluR subtype of the invention, or specific combinations of
subtypes. The resulting cell line can then be produced in amounts sufficient
for reproducible qualitative and quantitative analysis of the effects of a
receptor agonist, antagonist or allosteric modulator. Additionally, mRNA may
be produced by in vitro transcription of a DNA encoding a subtype of the
invention. This mRNA may be injected into Xenopus oocytes where the mRNA
directs the synthesis of the active receptor subtype. Alternatively, the
subtype-encoding DNA can be directly injected into oocytes. The transfected
mammalian cells or injected oocytes may then be employed in an drug
screening assay provided hereinafter. Such drugs are useful in diseases
associated with pathogenesis of a hmGluR subtype of the invention. Such
diseases include diseases resulting from excessive action of glutamate
preferentially mediated by hmGluRs, such as stroke, epilepsy and chronic
neurogenerative diseases. Particularly useful for assessing the specific
interaction of compounds with specific hmGluR subtypes are stably
transfected cell lines expressing a 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
hmGluR, said method comprising exposing cells containing heterologous DNA
encoding hmGluR of the invention, wherein said cells produce functional
hmGluR, to at least one compound or signal whose ability to modulate the
activity of said hmGluR is sought to be determined, and thereafter
monitoring said cells for changes caused by said modulation. Such an assay
enables the identification of agonists, antagonists and allosteric
modulators of a hmGluR of the invention.
In a further aspect, the invention relates to an assay for identifying
compounds which modulate the activity of a hmGluR subtype of the invention,
said assay comprising:
contacting cells expressing an active hmGluR subtype of the invention and
containing heterologous DNA encoding said hmGluR subtype with at least one
compound to be tested for its ability to modulate the activity of said
analysing cells for a difference in second messenger level or receptor
In particular, the invention covers an assay for identifying compounds which
modulate the activity of a hmGluR subtype of the invention, said assay
contacting cells expressing active hmGluR of the invention and containing
heterologous DNA encoding said hmGluR subtype with at least one compound to
be tested for its ability to modulate the activity of said receptor, and
monitoring said cells for a resulting change in second messenger activity.
The result obtained in the assay is compared to an assay suitable as a
Assay methods generally require comparison to various controls. A change in
receptor activity or in second messenger level is said to be induced by a
test compound if such an effect does not occur in the absence of the test
compound. An effect of a test compound on a receptor subtype of the
invention is said to be mediated by said receptor if this effect is not
observed in cells not expressing the receptor.
As used herein, a compound or signal that modulates the activity of a hmGluR
of the invention refers to a compound or signal that alters the response
pathway mediated by said hmGluR within a cell (as compared to the absence of
said hmGluR). A response pathway is activated by an extracellular stimulus,
resulting in a change in second messenger concentration or enzyme activity,
or resulting in a change of the activity of a membrane-bound protein, such
as a receptor or ion channel. A variety of response pathways may be
utilized, including for example, the adenylate cyclase response pathway, the
phospholipase C/intracellular calcium ion response pathway or coupling 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 cells expressing hmGluR of the invention may be employed for the
identification of compounds, particularly low molecular weight molecules
capable of acting as glutamate agonists or antagonists. Preferred are low
molecular weight molecules of less than 1,000 Dalton. Within the context of
the present invention, an agonist is understood to refer to a molecule that
is capable of interacting with a receptor, thus mimicking the action of
L-glutamate. In particular, a glutamate agonist is characterized by its
ability to interact with a hmGluR of the invention, and thereby increasing
or decreasing the stimulation of a response pathway within a cell. For
example, an agonist increases or decreases a measurable parameter within the
host cell, such as the concentration of a second messenger, as does the
natural ligand increase or decrease said parameter. For example, in a
suitable test system, wherein hmGluR of the invention is negatively coupled
to adenylate cyclase, e.g. CHO or BHK cells expressing a hmGluR of the
invention, such an agonist is capable of modulating the function of said
hmGluR such that the intracellular concentration of cAMP is decreased.
By contrast, in situations where it is desirable to tone down the activity
of hmGluR, antagonizing molecules are useful. Within the context of the
present invention, an antagonist is understood to refer to a molecule that
is capable of interacting with a receptor or with L-glutamate, but which
does not stimulate a response pathway within a cell. In particular,
glutamate antagonists are generally identified by their ability to interact
with a hmGluR of the invention, and thereby reduce the ability of the
natural ligand to stimule a response pathway within a cell, e.g. by
interfering with the binding of L-glutamate to a hmGluR of the invention or
by inhibiting other cellular functions required for the activity of hmGluR.
For example, in a suitable assay, e.g. an assay involving CHO or BHK cells
expressing a hmGluR subtype of the invention, a glutamate antagonist is
capable of modulating the activity of a hmGluR of the invention such that
the ability of the natural ligand to decrease the intracellular cAMP
concentration is weakened. Yet another alternative to achieve an
antagonistic effect is to rely on overexpression of antisense hmGluR RNA.
Preferred is an agonist or antagonist selectively acting on a receptor of
the hmGluR4 subfamily, e.g. hmGluR4, hmGluR6 or hmGluR7. Particularly useful
is an agonist or antagonist specifically modulating the activity of a
particular hmGluR subtype without affecting the activity of any other
An allosteric modulator of a hmGluR 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 allosteric modulator of a receptor of the invention. For
example, an allosteric modulator acting as agonist may enhance the specific
interaction between a 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 binding of the agonist is functionally
An in vitro assay for a glutamate agonist or antagonist may require that a
hmGluR of the invention is produced in sufficient amounts in a functional
form using recombinant DNA methods. An assay is then designed to measure a
functional property of the hmGluR protein, e.g. interaction with a
glutamatergic ligand. Production of a hmGluR of the invention is regarded as
occurring in sufficient amounts, if activity of said receptor results in a
For example, mammalian cells, e.g. HEK293 cells, L cells, CHO-K1 cells,
LLCPK-1 cells or GH3 cells (available e.g. from the American Tissue Type
Culture Collection) are adapted to grow in a glutamate reduced, preferably a
glutamate free, medium. A hmGluR expression plasmid, e.g. a plasmid
described in the Examples, is transiently transfected into the cells, e.g.
by calcium-phosphate precipitation (AusubeL F. M., et al. (1993) Current
Protocols in Molecular Biology, Greene and Wiley, USA). Cell lines stably
expressing a hmGluR of the invention may be generated e.g. by lipofectin-mediated
transfection with hmGluR expression plasmids and a plasmid comprising a
selectable marker gene, e.g. pSV2-Neo (Southern and Berg, J. Mol. Appl.
Genet. 1, 327-341 (1982)), a plasmid vector encoding the G418 resistence
gene. Cells surviving the selection are isolated and grown in the selection
medium. Resistant clonal cell lines are analyzed, e.g. for immunoreactivity
with subtype-specific hmGluR antibodies or by assays for hmGluR functional
responses following agonist addition. Cells producing the desired hmGluR
subtype are used in a method for detecting compounds binding to a hmGluR of
the invention or in a method for identifying a glutamate agonist or
In a further embodiment, the invention provides a method for identifying
compounds binding to a hmGluR subtype, said method comprising employing a
hmGluR subtype of the invention in a competitive binding assay. The
principle underlying a competitive binding assay is generally known in the
art. Briefly, binding assays according to the invention are performed by
allowing the compound to be tested for its hmGluR binding capability to
compete with a known, suitably labeled, glutamatergic ligand for the binding
site at the hmGluR target molecule. A suitably labeled ligand is e.g. a
radioactively labeled ligand, such as [3 H]glutamate, or a ligand which
can be detected by its optical properties, such as absorbance or
fluorescence. After removing unbound ligand and test compound the amount of
labeled ligand bound to hmGluR is measured. If the amount of labeled ligand
is reduced in the presence of the test compound this compound is said to be
bound to the target molecule. A competitive binding assay may be performed
e.g. with transformed or transfected host cells expressing a hmGluR of the
invention or a membraneous cellular fraction comprising a hmGluR of the
Compound bound to the target hmGluR may modulate the functional properties
of hmGluR and may thereby be identified as a glutamate agonist or antagonist
in a functional assay.
Functional assays are used to detect a change in the functional activity of
a hmGluR 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 hmGluR. A
functional response is e.g. a change (difference) in the concentration 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 a functional hmGluR of the invention (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 intracellular second
messenger indicative of the expression of an active hmGluR (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)), phosphatidyl inositol (PI) turnover assays
(Nakajima et al., J. Biol. Chem. 267, 2437-2442 (1992)), calcium ion flux
assays (Ito et al., J. Neurochem. 56, 531-540 (1991)), arachidonic acid
release assays (see, e.g. Felder et al., J. Biol. Chem. 264,20356-20362
(1989)), and the like.
More specifically, according to the invention a method for detecting a
glutamate agonist comprises the steps of (a) exposing a compound to a hmGluR
subtype of the invention 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 the hmGluR subtype, relative to
the absence of the tested compound and therefrom determining the presence of
a glutamate agonist.
A method for identifying a glutamate antagonist comprises the steps of (a)
exposing a compound in the presence of a known glutamate agonist to a hmGluR
subtype of the invention 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 compound with the hmGluR subtype,
relative to the stimulation of the response pathway by the glutamate agonist
alone and therefrom determining the presence of a glutamate antagonist
Inhibition may be detected, e.g. if the test compound competes with the
glutamate agonist for the hmGluR of the invention. Compounds which may be
screened utilizing such method are e.g. blocking antibodies specifically
binding to the hmGluR subtype. Furthermore, such an assay is useful for the
screening for compounds interacting with L-glutamate, e.g. soluble hmGluR
fragments comprising part or all of the ligand binding domain.
Preferentially, interaction of an agonist or antagonist with a hmGluR of the
invention denotes binding of the agonist or antagonist to said hmGluR.
As employed herein, conditions and times sufficient for interaction of a
glutamate agonist or antagonist candidate to the receptor will vary with the
source of the receptor, however, conditions generally suitable for binding
occur between about 4oC. and about 40oC., preferably between
about 4oC. and about 37oC., in a buffer solution between 0
and 2 M NaCl, preferably between 0 and 0.9 M NaCl, with 0.1 M NaCl being
particularly preferred, and within a pH range of between 5 and 9, preferably
between 6.5 and 8. Sufficient time for the binding and response will
generally be between about 1 ms and about 24 h after exposure.
Within one embodiment of the present invention, the response pathway is a
membrane-bound adenylate cyclase pathway, and, for an agonist, the step of
detecting comprises measuring a reduction or increase, preferably a
reduction, in cAMP production by the membrane-bound adenylate cyclase
response pathway, relative to the cAMP production in the relevant control
setup. For the purpose of the present invention, it is preferred that the
reduction or increase in cAMP production be equivalent or greater than the
reduction or increase induced by L-glutamate applied at a concentration
corresponding to its IC50 concentration. For an antagonist, the step of
detecting comprises measuring in the presence of the antagonist a smaller
L-glutamate induced decrease or increase in cAMP production by the
membrane-bound adenylate cyclase response pathway, as compared to the cAMP
production in the absence of the antagonist. The measurement of cAMP may be
performed after cell destruction or by a cAMP sensitive molecular probe
loaded into the cell, such as a fluorescent dye, which changes its
properties, e.g. its fluorescent properties, upon binding of cAMP.
Cyclic AMP production may be measured using methods well known in the art,
including for instance, methods described by Nakajima et al., supra, or
using commercially available kits, e.g. kits comprising radiolabeled cAMP,
e.g. [125 I]cAMP or [3 H]cAMP. Exemplary kits are the
Scintillation Proximity Assay Kit by Amersham, which measures the production
of cAMP by competition of iodinated-cAMP with cAMP antibodies, or the Cyclic
AMP [3 H] Assay Kit by Amersham.
In assay systems using cells expressing receptor subtypes that are
negatively coupled to the adenylate cyclase pathway, i.e. which cause a
decrease in cAMP upon stimulation and an increase in cAMP upon reduction of
stimulation, it is preferred to expose the cells to a compound which
reversibly or irreversibly stimulates the adenylate cyclase, e.g. forskolin,
or which is a phosphodiesterase inhibitor, such as isobutylmethylxanthine (IBMX),
prior to addition of the (potential) receptor agonist or antagonist.
Within another embodiment of the invention, the response pathway is the PI
hydrolysis/Ca2+ mobilization pathway. Such an assay for determining the
specific interaction of a test compound with a hmGluR subtype of the
invention may be functionally linked to changes in the intracellular calcium
ion (Ca2+) concentration. Several methods for determining a change in
the intracellular concentration of Ca2+ are known in the art, e.g. a
method involving a calcium ion sensitive fluorescent dye, such as fura-2
(see Grynkiewisz et al., J. Biol. Chem. 260, 3440-3450, 1985), fluo-3 or
Indo-1, such as the calcium fluor QuinZ method describe by Charest et al.
(J. Biol. Chem. 259, 8679-8773 (1993)), or the aequorin photoprotein method
described by Nakajima-Shimada (Proc. Natl Acad. Sci. USA 88, 6878-6882
(1991)). In one embodiment of the invention, intracellular calcium ion
concentration is measured by microfluoremetry in recombinant cells loaded
with calcium sensitive fluorescent dyes fluo-3 or fura-2. These measurements
may be performed using cells grown in a coverslip allowing the use of an
inverted microscope and video-imaging technologies or a fluorescence
photometer to measure calcium concentrations at the single cell level. For
both approaches, cells transformed with a hmGluR expressing plasmid have to
be loaded with the calcium indicator. To this end, the growth medium is
removed from the cells and replaced with a solution containing fura-2 or
fluo-3. The cells are used for calcium measurements preferentially during
the following 8 h. The microfluorometry follows standard procedures.
Ca2+ signals resulting from functional interaction of compounds with
the target molecule can be transient if the compound is applied for a
limited time period, e.g. via a perfusion system. Using transient
application several measurements can be made with the same cells allowing
for internal controls and high numbers of compounds tested.
Functional coupling of a hmGluR of the invention to Ca2+ signaling may
be achieved, e.g. in CHO cells, by various methods:
(i) coexpression of a recombinant hmGluR of the invention and a recombinant
voltage-gated cation channel, activity of which is functionally linked to
the activity of the hmGluR;
(ii) expression of a chimeric hmGluR receptor, which directly stimulates the
(iii) coexpression of a recombinant hmGluR of the invention with a
recombinant Ca2+ -permeable cAMP dependent cation channel.
In other expression systems functional coupling of a hmGluR to Ca2+
signalling may be achieved by transfection of a hmGluR of the invention if
these cells naturally express (i) voltage gated Ca channels, activity of
which is functionally linked to activity of mGluRs or (ii) Ca2+
-permeable cAMP dependent ion channels. For example, GH3 cells which
naturally express voltage-gated Ca channels, directly allow application of
Ca2+ assays to test for hmGluR functional activity by cotransfection of
Further cell-based screening assays can be designed e.g. by constructing
cell lines in which the expression of a reporter protein, i.e. an easily
assayable protein, such as .beta.-galactosidase, chloramphenicol
acetyltransferase (CAT) or luciferase, is dependent on the 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 comprising the enzyme DNA is stably transfected into
a host cell. The host cell is then transfected with a second DNA construct
containing a first DNA segment encoding a hmGluR of the invention operably
linked to additional DNA segments necessary for the expression of the
receptor. For example, if binding of a test compound to the hmGluR of the
invention results in elevated cAMP levels, the expression of luciferase is
induced or decreased, depending on the promoter chosen. The luciferase is
exposed to luciferin, and the photons emitted during oxidation of luciferin
by the luciferase is measured.
The drug screening assays provided herein will enable identification and
design of receptor subtype-specific compounds, particularly ligands binding
to the receptor protein, eventually leading to the development of a
disease-specific drug. If designed for a very specific interaction with only
one particular hmGluR subtype (or a predetermined selection of hmGluR
subtypes) such a drug is most likely to exhibit fewer unwanted side effects
than a drug identified by screening with cells that express a(n) (unknown)
variety of receptor subtypes. Also, testing of a single receptor subtype of
the invention or specific combinations of different receptor subtypes with a
variety of potential agonists or antagonists provides additional information
with respect to the function and activity of the individual subtypes and
should lead to the identification and design of compounds that are capable
of very specific interaction with one or more receptor subtypes.
In another embodiment the invention provides polyclonal and monoclonal
antibodies generated against a hmGluR subtype of the invention. Such
antibodies may useful e.g. for immunoassays including immunohistochemistry
as well as diagnostic and therapeutic applications. For example, antibodies
specific for the extracellular domain, or portions thereof, of a particular
hmGluR subtype can be applied for blocking the endogenous hmGluR subtype.
The antibodies of the invention can be prepared according to methods well
known in the art using as antigen a hmGluR subtype of the invention, a
fragment thereof 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 distinguishing between the active or inactive
form. Factors to consider in selecting subtype fragments as antigens (either
as synthetic peptide or as fusion protein) include antigenicity,
accessibility (i.e. extracellular and cytoplasmic domains) and uniqueness to
the particular subtype.
Particularly useful are antibodies selectively recognizing and binding to
receptor subtypes of the above described subfamily without binding to a
subtype of another subfamily and antibodies selectively recognizing and
binding to one particular subtype without binding to any other subtype.
The antibodies of the invention can be administered 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
Claim 1 of 5 Claims
What is claimed is:
1. A purified human metabotropic glutamate receptor comprising an amino acid
sequence as set forth in SEQ ID NO: 12.
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