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WO1995022609A2 - Proteines des recepteurs humains du glutamate - Google Patents

Proteines des recepteurs humains du glutamate Download PDF

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Publication number
WO1995022609A2
WO1995022609A2 PCT/GB1995/000356 GB9500356W WO9522609A2 WO 1995022609 A2 WO1995022609 A2 WO 1995022609A2 GB 9500356 W GB9500356 W GB 9500356W WO 9522609 A2 WO9522609 A2 WO 9522609A2
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leu
human
ser
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gly
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PCT/GB1995/000356
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WO1995022609A3 (fr
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Andrew Joseph Makoff
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The Wellcome Foundation Limited
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Priority claimed from GB9403285A external-priority patent/GB9403285D0/en
Priority claimed from GB9415532A external-priority patent/GB9415532D0/en
Application filed by The Wellcome Foundation Limited filed Critical The Wellcome Foundation Limited
Publication of WO1995022609A2 publication Critical patent/WO1995022609A2/fr
Publication of WO1995022609A3 publication Critical patent/WO1995022609A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the novel human metabotropic glutamate receptors 3 and 4 (human mGluR3 and human mGluR4) and polypeptide fragments thereof, DNA coding therefor and oligonucleotide fragments thereof, antibodies specific for the receptors and a method for screening for compounds capable of binding to or modulating the activity of the receptors.
  • L-glutamate serves as a major excitatory neurotransmitter.
  • the interaction of glutamate with its membrane bound receptors is believed to play a role in many important neuronal processes including fast synaptic transmission, synaptic plasticity, and long term potentiation. These processes are fundamental to the maintenance of life and normal human abilities such as learning and memory (Monaghan, D.T. et al. , 8 Neuron 267 (1992)) .
  • L-glutamate In addition to its role in normal human physiology, interaction of L-glutamate with its receptors is believed to play a key role in many neurological disorders such as stroke, epilepsy, and head trauma, as well as neurodegenerative processes such as
  • Alzheimer' s disease (Olney, R.W. , 17 Drug Dev.Res. 299 (1989)) .
  • rat mGluRs Pharmacological characterisation of receptors for L-glutamate has led to their classification into two families based on their biological function: the ionotropic receptors which.are directly coupled to cation channels in the cell membrane, and the metabotropic receptors which function through coupling to G-proteins.
  • rat mGluRs seven different rat mGluRs and three different human mGluRs have been cloned. These are: rat mGluRl, (Masu et al, Nature, Vol 349, 760 - 765, (1991) , Houamed, K.M., et al . Science, Vol 252, 1318-1321, (1991)) ; rat mGluR2, (Tanabe, Y. et al.
  • the other five mGluRs are all negatively coupled to adenylate cyclase (Nakanishi, S., Science 258, 597-603, (1992); Nakajima, Y. , Iwakabe, H. , Akazawa, C, Nawa, H., Shigemoto, R. , Mizuno, N. and Nakanishi, S., J. Biol. Chem. 268, 11868-11873 (1993) ; Okamoto, N. et al, J. Biol. Chem. vol 269, 1231-1236 (1994) ; Saugstad, J.A. et al, Mol. Pharmacol, vol 45, 367-372 (1994)) .
  • mGluRl and 5 (guisqualate)
  • mGluR2 and 3 [(2S,1'R,2'R,3'R)2- (2,3-dicarboxycyclopropyl)glycine, DCG-IV]
  • mGluR4, 6 and 7 (2-amino-4-phosphonobutyrate, AP4)
  • Nakanishi, S., Science 258, 597-603, (1992) Nakajima, Y. , Iwakabe, H., Akazawa, C, Nawa, H. , Shigemoto, R.
  • the present invention provides a DNA isolate encoding human mGluR3 or human mGluR4.
  • the DNA isolate may encode human mGluR3 having an amino acid sequence at least 97% (e.g. at least 98% or at least 99%) identical to the sequence of SEQ ID NO:2.
  • the DNA isolate may alternatively encode human mGluR4 having an amino acid sequence at least 97% (e.g. at least 98% or at least 99%) identical to the sequence of SEQ ID NO:4.
  • the DNA isolate encodes human mGluR3 having exactly the amino acid sequence of SEQ ID NO:2 or human mGluR4 having exactly the amino acid sequence of SEQ ID NO:4.
  • the nucleotide sequence of the DNA isolate may be at least 90% identical (e.g. at least 95%, at least 96%, at least 97% at least 98% or at least 99% identical) to the human mGluR3 coding sequence set forth from position 253 or 259 to position 2889 of SEQ ID NO:l.
  • the nucleotide sequence may alternatively be at least 90% identical (e.g. at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical) to the human mGluR4 coding sequence set forth from position 171 to position 2906 of SEQ ID NO:3.
  • nucleotide sequence of the DNA isolate is exactly the sequence from position 253 or 259 to position 2889 of SEQ ID NO:l or exactly the nucleotide sequence from position 171 to position 2906 of SEQ ID NO:3.
  • these sequences from SEQ ID NOs:3 and 4 may be changed by nucleotide substitution, deletion, insertion or extension.
  • Such changed sequences should still encode a protein which retains mGluR3 or mGluR4 activity, e.g. L-glutamate binding and/or negative coupling to adenylate cyclase.
  • a substitution, deletion or insertion may involve one or more nucleotides, typically from one to five, one to ten or one to twenty nucleotides.
  • the human mGluR3 nucleotide and amino acid sequences set forth in present SEQ ID NO:l contain three differences compared to the human mGluR3 sequences set forth in WO 94/29449 (see SEQ ID NO:5) , as follows:
  • GGC in the WO 94/29449 sequence is Asp (GAC) in the present sequence and Glu 374 (GAA) in the WO 94/29449 sequence is Asp (GAC) in the present sequence.
  • the mGluR3 nucleotide sequence contains two ATG codons in the same reading frame at the beginning of the translated sequence.
  • the earlier of the two codons is assumed to be the start codon so the translated sequence begins MetLysMet....
  • the sequence surrounding the second ATG codon is much more favourable for initiation of translation and it is therefore likely that this codon initiates translation giving a translated sequence which begins MetLeuThr.... It is possible that two protein products are produced from the two initiation codons and both products are included in the invention.
  • the invention includes an oligonucleotide fragment having a sequence of a portion of a DNA isolate encoding human mGluR3 or mGluR4 of the invention.
  • the fragment may have a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to sequence encoding human mGluR3 or human mGluR4 of the invention, for example sequence shown ih SEQ ID NO:l or SEQ ID NO:3.
  • the fragment is at least 12 nucleotides in length, e.g. at least 15, at least 18, at least 30, at least 60, at least 180, at least 360 or at least 720 nucleotides in length.
  • the fragment may be single or double stranded.
  • the fragment When the fragment is single stranded, it may have a sequence from either a sense or antisense strand of a DNA molecule encoding a human mGluR3 or a human mGluR4 of the invention. .An antisense fragment may be useful in the therapeutic treatment of a disease involving over-expression of human mGluR3 or human mGluR4.
  • the present invention also provides a human mGluR3 protein and a human mGluR4 protein, such as those having the amino acid sequences of SEQ ID NOs:2 and 4.
  • a human mGluR3 or mGluR4 protein having a sequence which is at least 96% identical to the amino acid sequence of SEQ ID NO:2 or 4, for example a sequence at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 2 or 4.
  • the invention further includes a polypeptide fragment of a human mGluR3 or mGluR4 protein of the invention, which fragment is at least 8 amino acids in length, for example at least 12, at least 24, at least 48 or at least 96 amino acids in length.
  • the human mGluR3 or mGluR4 protein will usually be obtained by recombinant DNA techniques. However, the protein may be obtained using biochemical purification of the protein from its natural origin.
  • the human mGluR3 or mGluR4 is generally expressed on the surface of a host cell, but may be in purified or isolated form.
  • the mGluR3 or mGluR4 in purified form comprises a preparation in which at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the weight of protein in the preparation is human mGluR3 or mGluR4.
  • a DNA isolate encoding a fragment of the amino acid sequence of the human mGluR3 or mGluR4 protein.
  • DNA sequences encoding fragments of the human mGluR3 or mGluR4 protein preferably encode those parts of the amino acid sequence which characterise the receptor, i.e. those parts which are most distinct from other human mGlu receptor proteins.
  • a person of ordinary skill in the art would by reference to the sequences disclosed herein know how to obtain a DNA isolate according to the invention using methodologies and techniques well known to the skilled person.
  • the various means include for example, DNA synthesis, or more preferably, recombinant DNA techniques .
  • Techniques for constructing recombinant isolates are described by Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) .
  • Human mGluR3 or mGluR4 protein and fragments thereof may be obtained by expression of DNA isolates of the present invention in appropriate expression vectors.
  • a library of replicable expression vectors may be created by cloning genomic DNA or, more preferably, cDNA into a parent vector. The library is screened for clones containing the desired nucleic acid sequence, e.g. by means of a DNA probe. Probes having consensus sequences for specific regions of various human mGluR proteins may be used. For example, the following consensus primers designed from the published sequences of rat mGluRl to 5 proteins may be used:
  • ACAACGACACACCCGTGGTCAA SEQ ID NO:5
  • TCCGGTCGGGAGCTCTGCTA SEQ ID NO:6
  • GCTACTCTGCCCTGCTGACCAAGAC SEQ ID NO:7
  • GCAATGCGGTTGGTCTTGGT SEQ ID NO:8
  • AAGTTCATCGGCTTCACCATGTACAC SEQ ID NO:9
  • a replicable expression vector is a vector which contains the appropriate origin of replication sequence for replication of the vector and the appropriate sequences for expression of the foreign nucleotide sequence in the vector.
  • the sequences for expression of a foreign sequence will generally include a transcription promoter operably linked to the foreign sequence.
  • operably linked refers to a linkage in which the promoter and foreign sequence are connected in such a way to permit expression of the foreign sequence.
  • the transcription promoter sequence may be part of the parent vector sequence into which the foreign sequence is inserted.
  • the promoter sequence may be a native promoter sequence of a gene encoding human mGluR3 or mGluR4 of the invention.
  • a vector may be, for example, a plasmid, virus or phage vector.
  • a vector may contain one or more selectable markers, for example an ampicillin resistance gene in the case of a bacterial vector or a neomycin resistance gene in the case of a mammalian vector.
  • a foreign gene sequence inserted into a vector may be transcribed in vitro or the vector may be used to transform or transfect a host cell.
  • a host cell transformed or transfected with a vector into which there has been inserted a DNA isolate (or a fragment thereof) encoding human mGluR3 or mGluR4 protein.
  • a vector and host cell will be chosen so as to be compatible with each other, and may be prokaryotic or eukaryotic.
  • a prokaryotic host may, for example, be E. coli, in which case the vector may, for example, be a bacterial plasmid or a phage vector.
  • a eukaryotic host may, for example, be a yeast (e.g.
  • S.cerevisiae cell, a Chinese hamster ovary (CHO) cell, BHK cell, oocyte or an insect cell (e.g. Spodoptera frugiperda) .
  • insect cell e.g. Spodoptera frugiperda
  • the vector is generally a baculovirus vector (reviewed by Luckow and Summers in BIO/TECHNOLOGY, Vol. 6, 47-55 (1988)) .
  • the host is an oocyte mRNA encoding human mGluR3 or mGluR4 protein can be injected into the oocyte for expression of the protein.
  • a host cell expressing human mGluR3 or a human mGluR4 according to the invention may be produced by a method comprising
  • An oligonucleotide fragment according to the invention will generally be DNA, although other types of nucleic acid may be used, for example RNA or modified DNA.
  • a number of different types of nucleic acid modification are known in the art . These include methylphosphonate and phosphorothioate backbones, and addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • Oligonucleotide fragments may be useful in the detection of nucleic acid sequences.
  • An oligonucleotide fragment corresponding to a portion of the human mGluR3 or mGluR4 coding sequence may be an oligonucleotide probe or an oligonucleotide primer (e.g. a polymerase chain reaction (PCR) primer) , which will hybridise to a nucleic acid molecule (e.g. a DNA or RNA molecule) encoding a human mGluR3 or mGluR4 protein of the invention.
  • the probe or a pair of primers may be used to detect or quantitatively determine the nucleic acid sequence. This has diagnostic utility in detecting and quantitatively determining human mGluR3 or mGluR4 mRNA associated with a disease state, for example disorders of the central nervous system.
  • a fragment which is a probe or primer may carry a revealing label, such as 32P, digoxigenin or biotin.
  • the probe or primer will specifically hybridise only to its target sequence, e.g. a portion of the sequence disclosed in SEQ ID NO:l or SEQ ID NO:3, and not to other sequences.
  • a probe or primer which hybridises only to its target sequence will generally be exactly complementary to the target sequence whereas a probe or primer which is only selective may have a sequence which is, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% complementary to the target sequence.
  • a probe which is not exactly complementary to its target sequence has utility in the identification of new human mGluR coding nucleotide sequences.
  • a fragment which is a probe or primer may have from 12 to 60 nucleotides, e.g. from 12 to 40 nucleotides, or from 15 to 30 nucleotides.
  • Primers for PCR are generally provided as a pair.
  • a first primer hybridises to a sense sequence 3' to the sequence to be amplified and a second primer hybridises to an antisense sequence 5' to the sequence to be amplified. This allows synthesis of double stranded DNA representing the region between the two primers.
  • the invention provides a method of amplifying a target nucleic acid sequence present in a nucleic acid encoding a human mGluR3 or mGluR4 protein of the invention, which method comprises carrying out PCR employing a primer of the invention.
  • Such a method generally comprises carrying out cycles of
  • the number of cycles is suitably from 10 to 50, preferably 20 to 40, more preferably 25 to 35.
  • the method may be carried out starting from a double stranded nucleic acid (e.g. dsDNA) or a single stranded nucleic acid (e.g. mRNA) .
  • the target sequence may be a complete human mGluR3 or mGluR4 protein encoding sequence or a partial human mGluR3 or mGluR4 protein encoding sequence.
  • PCR polymerase chain reaction
  • An oligonucleotide probe according to the invention has utility in detecting or quantitatively determining a nucleic acid (e.g. a DNA or RNA) encoding a human mGluR3 or mGluR4 protein according to the invention.
  • a nucleic acid e.g. a DNA or RNA
  • Conventional methods for detecting or quantitatively determining a nucleic acid may be used, for example in situ hybridization, Southern blotting or Northern blotting. Accordingly, there is provided a method of detecting or quantitatively determining in a sample a target nucleic acid encoding human mGluR3 or mGluR4 according to the invention, which method comprises
  • the sample containing the target nucleotide sequence may, for example, be a tissue specimen, a tissue extract or cell extract from a patient suffering from a disease associated with abnormal human mGluR3 or mGluR4 protein activity such as a disorder of the central nervous system.
  • the sample may, for example, be a sample produced as a result of a recombinant DNA procedure, in which case the sample may be recombinant cells.
  • the target nucleic acid sequence may be a complete human mGluR3 or mGluR4 protein coding sequence or a partial human mGluR3 or mGluR4 protein coding sequence.
  • a preferred method of detecting or quantitatively determining a target nucleic acid sequence in a sample comprises
  • a probe can be used in an in situ hybridization procedure to locate a nucleic acid sequence encoding a human mGluR3 or mGluR4 protein of the invention. This can be done to determine the spatial distribution of human mGluR3 or mGluR4 protein coding mRNA sequences in a cell or tissue. For mRNA detection, the tissue is gently fixed so that its RNA is retained in an exposed form and the tissue is then incubated with a labelled complementary probe.
  • the invention includes an antibody specific for human mGluR3 or mGluR4 protein according to the invention.
  • the antibody has utility in detecting and quantitatively determining human mGluR3 or mGluR4 protein, and hence is useful in diagnosis of diseases associated with human mGluR3 or mGluR4 protein, such as the diseases listed hereinabove.
  • the antibody also has utility in production of human mGluR3 or mGluR4 protein by recombinant DNA procedures, for example in detection of positive clones containing a target sequence.
  • the antibody is preferably monoclonal, but may also be polyclonal.
  • the antibody may be labelled.
  • suitable antibody labels include radiolabels, biotin (which may be detected by avidin or streptavidin conjugated to peroxidase) , alkaline phosphatase and fluorescent labels (e.g. fluorescein and rhodamine) .
  • the term "antibody” is used herein to include both complete antibody molecules and fragments thereof. Preferred fragments contain at least one antigen binding site, such as Fab and F(ab')2 fragments. Humanised antibodies and fragments thereof are also included within the term "antibody” .
  • the antibody is produced by raising antibody in a host animal against a human mGluR3 or mGluR4 protein according to the invention or an antigenic epitope (e.g. a peptide) thereof
  • the immunogen (hereinafter "the immunogen") .
  • a method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.
  • a method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein, Nature 256, 495-497, (1975)) .
  • the antibody may also be produced by recombinant DNA technology, for example as described in Skerra et al (1988) Science 240 , 1038-1041.
  • An immortalized cell producing the desired antibody may be selected by a conventional procedure.
  • the hybridomas may be grown in culture or injected mtraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host.
  • Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.
  • the experimental animal is suitably a goat, rabbit, rat or mouse.
  • the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitably carrier.
  • the carrier molecule is typically a physiologically acceptable carrier.
  • the antibody obtained may be isolated and, if desired, purified.
  • the invention provides a method of detecting or quantitatively determining in a sample a human mGluR3 or mGluR4 protein of the invention, which method comprises
  • a preferred method for detecting or quantitatively determining a human mGluR3 or mGluR4 protein is Western blotting. Such a method can comprise the steps of
  • ELISA enzyme-linked immunoassay
  • non-competitive ELISA methods Preferred methods of quantitative determination are ELISA (enzyme-linked immunoassay) methods such as non-competitive ELISA methods.
  • an ELISA method comprises the steps of
  • an antibody of the invention may be employed histologically for in situ detection of a human mGluR3 or mGluR4 protein, e.g. by immunofluorescence or immunoelectron micropsy. In situ detection may be accomplished by removing a histological specimen from a patient, and allowing a labelled antibody to bind to the specimen. Through use of such a procedure, it is possible to determine not only the presence of a human mGluR3 or mGluR4 protein but also its distribution.
  • an antibody of the invention may be used to purify a target human mGluR3 or mGluR4 protein.
  • Conventional methods of purifying an antigen using an antibody may be used. Such methods include immunoprecipitation and immunoaffinity column methods.
  • an antibody in accordance with the invention is coupled to the inert matrix of the column and a sample containing the target human mGluR3 or mGluR4 protein is passed down the column, such that the target human mGluR3 or mGluR4 protein is retained. The human mGluR3 or mGluR4 protein is then eluted.
  • the sample containing the target mGluR3 or mGluR4 protein used in the detection, determination and purification methods may be a tissue specimen, a tissue extract or a cell extract from a patient suffering from a disease associated with human mGluR3 or mGluR4 protein such as a disease listed hereinabove.
  • the sample may be one produced as a result of recombinant DNA procedures, e.g. an extract of host cells.
  • the invention provides a pharmaceutical composition containing a pharmaceutically acceptable carrier or diluent and, as active ingredient, an antibody or polypeptide fragment of the invention.
  • a human mGluR3 or mGluR4 protein of the invention is useful for screening for drugs which modulate (e.g. inhibit or stimulate) the receptor.
  • the invention includes a method for identifying a compound which binds to or modulates the activity of the human mGluR3 or mGluR4 protein, which method comprises one or more of the following steps:
  • the invention also extends to agents (compounds) identified by use of a particular screen.
  • the agent is a chemical molecule of relatively low molecular weight, for example less than about 1000D.
  • Methods for identifying compounds which bind to human mGluR3 or human mGluR4 receptor generally comprise the following steps : (a) contacting a compound with the receptor, and
  • a competitive binding assay may, for example, be used.
  • a competitive binding assay may comprise
  • step (c) comparing the amount of the labelled ligand bound to the receptor measured in step (b) with the amount of the labelled ligand that binds to the receptor in the absence of the test compound.
  • step (b) If less labelled ligand binds to the receptor in the presence of the test compound than in the absence of the test compound, this indicates that the test compound is competing for binding to the receptor with the ligand.
  • the amount of the ligand bound to the receptor in step (b) may be measured indirectly; the amount of bound ligand may be determined by extrapolation from the amount of unbound ligand.
  • Methods for identifying compounds which modulate the activity of human mGluR3 or human mGluR4 receptor generally comprise
  • the second messenger activity measured in step (b) may be basal or stimulated adenylate cyclase activity.
  • Adenylate cyclase activity may be measured by measuring the level of cAMP, which is the product of adenylate cyclase.
  • cAMP the product of adenylate cyclase.
  • adenylate cyclase is stimulated using forskolin and any decrease in adenylate cyclase activity caused or prevented by the presence of the test compound is measured.
  • a suitable adenylate cyclase assay is described in Tanabe et al (1992) Neuron 8 . , 169-179.
  • the invention includes a combination (or complex) of human mGluR3 or human mGluR4 receptor and an agent (compound) which modulates the second messenger activity of the receptor.
  • agents are L-glutamate (mGluR3 and mGluR4) ; (2S, l'R,2'R,3'R) 2- (2, 3-dicarboxycyclopropyl)glycine, DCG-IV (mGluR3) ; L-2-amino-4-phosphonobutyrate, L-AP4 (mGluR4) ; trans- l-aminocyclopentane-l,3-dicarboxylate, t-ACPD (mGluR4) ; and quisqualate (mGluR4) .
  • the invention also includes a method of modulating the second messenger activity of human mGluR3 or human mGluR4 receptor, which method comprises contacting the receptor with a compound which modulates the second messenger activity of the receptor. Examples of suitable compounds which modulate receptor activity are given above.
  • the method may be carried out either on the human/animal body or outside the human or animal body (e.g. in vitro) .
  • Figure 1 shows a comparison between human and rat mGluR3 protein sequences.
  • the human amino acid sequence (SEQ ID NO:2) is given with differences in the rat sequence shown underneath.
  • the seven putative transmembrane domains (TMD-I to VII) are underlined.
  • FIG. 2 shows a comparison between human and rat mGluR4 protein sequences.
  • the human amino acid sequence (SEQ ID NO:4) is given with differences in the rat sequence shown underneath.
  • the seven putative transmembrane domains (TMD-I to VII) are underlined.
  • Figure 3 shows the effect of mGluR4 ligands on the accumulation of forskolin-stimulated cyclic AMP.
  • Dose response curves are given for L-amino-4-phosphonobutyrate (L-AP4) , glutamate, trans-1-aminocyclopentane-l, 3-dicarboxylate (t-ACPD) and quisqualate.
  • Human brain material from the median frontal cortex and amygdala from one individual was used as the source of mRNA using the Fast Track mRNA Isolation Kit (Invitrogen) .
  • the mRNA was used to construct a library containing a mixture of oligo-dT and random hexamer-primed cDNA, using the Timesaver cDNA Synthesis Kit (Pharmacia) and ⁇ ZAP II arms (Stratagene) .
  • PCR polymerase chain reaction
  • a computer-generated alignment of rat sequences of mGluR cDNAs 1 to 5 revealed a number of regions of high homology. Seven regions were used to design oligonucleotide primers such that
  • each primer contained a consensus sequence for all five genes, (b) the highest homology of each primer was towards its 3' end and (c) the most 3' base was never a mismatch.
  • Four primers (BN29, 30, 31 and 33) were from the coding strand and three primers (BN32, 34 and 35) were from the other strand. All seven primers came from the region of each gene which encodes the seven transmembrane domains.
  • PCR amplifications were performed with all nine combinations of the primers using either human genomic or human brain cDNA. Analysis of these PCR products showed that with several primer pairs, more than one mGluR subtype was amplified. Between the primer pairs all five human genes homologous to the five rat mGluR protein genes used to generate the primers were represented (Table 1) . There was generally a strong bias in favour of the human mGluR3 gene. Since mGluR3 was also over-represented in PCRs involving genomic DNA, it may reflect a bias in the primers rather than the relative abundance of mGluR3 mRNA in the brain samples. When the annealing temperature in the PCR conditions was lowered to 38°C the distribution of mGluRs among the clones analysed was more even.
  • PCR conditions were: 35 cycles of 96°C for 35 sec, 56°C for 2 min and 72°C for 2 min.
  • the PCR products were separated by electrophoresis, the bands excised and the isolated fragments cloned into pT7Blue T-vector (Novagen) .
  • the products were identified by sequencing of the cloned fragments.
  • the cDNA library was screened using a mixed probe derived from inserts from cloned PCR products specific to each of human mGluRl to 5. Hybridization was at 65°C in 10% dextran sulphate, 1% SDS, 0.1% sodium pyrophosphate, 1M Tris-Cl (pH 7.5) and lOO ⁇ g/ml single stranded salmon testis DNA. The filters were washed in 1 x SSC and 1% SDS at 65oC followed by 0.1 x SSC at room temperature. Three phage clones were isolated and stored in SM buffer (Sambrook et al, ibid) . The largest of these extended from the 3' end to within 500 bp of the initiating ATG codon.
  • PCR on the amplified phage was repeated but with samples of increasing dilution until the signal was lost. From this titration, it could be calculated that the PCR signal was derived from approximately 1 per 10 5 phage, consistent with one positive phage on the original plate of approximately 80,000 independent clones.
  • the positive clone was recovered from the amplified mixture using PCR after each of several rounds of dilution and replication as described by Israel , D.I., Nucl. Acids Res., 21, 2627-2631, (1993) .
  • This fourth clone was found to contain the complete coding sequence of human mGluR3. The sequence was determined for both strands of the entire insert and is shown in SEQ ID N0:1.
  • Plasmids containing the inserts were excised using helper phage R408 (Stratagene) . Some sequencing was performed on such plasmid clones using flanking or internal primers. Most was on sub-clones generated by limited exonuclease III digestion using the Nested Deletion Kit (Pharmacia) .
  • a human cDNA library was constructed in the same way as Example 1, except that human brain material from the cerebellum was used as the source of mRNA.
  • Source of Probes
  • the cDNA library was screened using an mGluR4 probe derived from inserts from cloned PCR products specific to human mGluR4.
  • Hybridization was at 65°C in 10% dextran sulphate, 1% SDS, 0.1% sodium pyrophosphate, 1M Tris-Cl (pH 7.5) and 100 ⁇ g/ml single stranded salmon testis DNA.
  • the filters were washed in 1 x SSC and 1% SDS at 65°C followed by 0.1 x SSC at room temperature.
  • Phage clones were stored in SM buffer (Sambrook et al, ibid) .
  • New PCR primers (AP43 and AP44) (see herebelow) were designed from the published rat mGlu4 sequence. These were located in order to generate a product immediately downstream of the start of the coding sequence.
  • Another clone was recovered from the same cerebellum cDNA library using PCR using the new primers (AP43 and AP44 see herebelow) after each of several rounds of dilution and replication as described by Israel, D.I., Nucl. Acids Res., 21, 2627-2631, (1993) .
  • This second clone (clone 31) was found to contain the 5' end of the coding sequence of human mGluR4, but did not overlap the 3' clone (clone 17) .
  • Another pair of primers (CA396 and 400) were designed using the 3' end of clone 31 and the rat sequence in the gap between clones 31 and 17.
  • the primers AP47 and AP48 were designed for this purpose.
  • DHfr CHO cells were grown at 37°C in an atmosphere containing 5% C0 2 in glutamate-free Dulbecco's modified Eagles medium supplemented with 5% dialysed foetal calf serum, 2mM glutamine, ImM proline, lOO ⁇ M hypoxanthine and 16 ⁇ M thymidine. Petri dishes (10cm) were seeded with 10 6 cells and incubated for 24 hours. The cells were washed with serum-free medium and replaced by fresh serum-free medium containing lO ⁇ g pSVLHMGR3 and l ⁇ g pSV2 dhfr [Subramani, S. et al. Mol. Cell. Biol.
  • Forskolin-stimulated cyclic AMP assay Approximately 2 x 10 5 cells were seeded in each well of a 12 well microtitre plate. After incubation at 37°C for 24 hours the cells were exposed to 100 ⁇ 3-isobutyl-l-methylxanthine (phosphodiesterase inhibitor) at 37°C for 20 minutes followed by both the ligand and lO ⁇ M forskolin for 10 minutes. The medium was removed and ice-cold ethanol was added to the cells. After 2 hours at room temperature the supernatant was transferred to fresh tubes and the ethanol was removed by evaporation. The accumulation of cyclic AMP was measured in each tube using a scintillation proximity assay (Amersham) .
  • the complete mGluR4 coding sequence was constructed from clones 17, 31 and 32, utilising the restriction endonucleases EcoRI (SEQ ID NO:3, position 290-295) , Fspl (SEQ ID NO:3, position 1615-1620) and Ncol (SEQ ID N0:3, position 2915-2920) .
  • a unique BamHI site was introduced downstream of both the TAG stop codon and of the Ncol site by subcloning the EcoRI-FspI fragment from clone 32 and the Fspl-Ncol fragment from clone 17 into pSL1190 (Pharmacia) .
  • the sequence TCTAGG in clone 31 (SEQ ID NO:3, positions 155-160) upstream of the initiating ATG codon was mutated to TCTAGA (Xbal recognition sequence) by PCR using primers CA587 and CA565.
  • the entire coding sequence was cloned as an Xbal-BamHI fragment into the expression vector pSVL (Pharmacia) in which transcription of mGluR4 is under the control of the SV40 late promoter.
  • the resulting vector is pSVLHMGR4.
  • Dhrf CHO cells were grown at 37°C in an atmosphere containing 5% C0 2 in glutamate-free Dulbecco's modified Eagles medium supplemented with 5% dialysed foetal calf serum, 2mM glutamine, ImM proline, lOO ⁇ M hypoxanthine and 16 ⁇ M thymidine. Petri dishes (10cm) were seeded with 10 6 cells and incubated for 24 hours. The cells were washed with serum-free medium and replaced by fresh serum-free medium containing lO ⁇ g pSVLHMGR4 plus pSV2 dhfr [Subramani S. et al Mol. Cell. Biol. Vol 1, 854
  • AAAGCATCTG TA ⁇ AATGTA AAGATACTGA GAATAAAACC ⁇ CAAGG ⁇ T T 3410

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Abstract

L'invention concerne les récepteurs 3 et 4 métabotropiques humains du glutamate (mGluR3 et mGluR4 humains), les ADN codant pour ces récepteurs ainsi qu'un procédé permettant de trier les composés qui se fixent aux récepteurs ou bien qui en modifient l'activité.
PCT/GB1995/000356 1994-02-21 1995-02-21 Proteines des recepteurs humains du glutamate WO1995022609A2 (fr)

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GB9403285A GB9403285D0 (en) 1994-02-21 1994-02-21 Human glutamate receptor protein
GB9403285.1 1994-02-21
GB9415532A GB9415532D0 (en) 1994-08-01 1994-08-01 Human glutamate receptor protein
GB9415532.2 1994-08-01

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WO1995022609A2 true WO1995022609A2 (fr) 1995-08-24
WO1995022609A3 WO1995022609A3 (fr) 1995-09-08

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996029404A1 (fr) * 1995-03-20 1996-09-26 Sibia Neurosciences, Inc. SOUS-TYPE mGluR6 DE RECEPTEUR METABOTROPE DE GLUTAMATE CHEZ L'HOMME
WO1999021992A3 (fr) * 1997-10-23 1999-09-10 Ganimed Pharmaceuticals Gmbh Molecules d'acides nucleiques codant pour un recepteur de glutamate
US6228610B1 (en) 1993-09-20 2001-05-08 Novartis Corporation Human metabotropic glutamate receptor subtypes (hmR4, hmR6, hmR7) and related DNA compounds
US6384205B1 (en) * 1996-03-12 2002-05-07 Eli Lilly And Company Metabotropic glutamate receptor 4 nucleic acid
WO2005069010A1 (fr) * 2004-01-19 2005-07-28 Bayer Healthcare Ag Diagnostics et traitements de pathologiques associees au recepteur metabotropique du glutamate 4 (mglur4) humain
US7262280B1 (en) 1998-04-03 2007-08-28 Nps Pharmaceuticals, Inc. G-protein fusion receptors and constructs encoding same
US11254744B2 (en) 2015-08-07 2022-02-22 Imaginab, Inc. Antigen binding constructs to target molecules
US11266745B2 (en) 2017-02-08 2022-03-08 Imaginab, Inc. Extension sequences for diabodies

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ240921A (en) * 1990-12-12 1994-06-27 Zymogenetics Inc G protein coupled glutamate receptor (neurotransmitters), recombinant production
US5521297A (en) * 1993-06-04 1996-05-28 Salk Institute Biotechnology/Industrial Associates Nucleic acids encoding human metabotropic glutamate receptors
US6228610B1 (en) * 1993-09-20 2001-05-08 Novartis Corporation Human metabotropic glutamate receptor subtypes (hmR4, hmR6, hmR7) and related DNA compounds

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5912122A (en) * 1993-06-04 1999-06-15 Sibia Neurosciences, Inc. Nucleic acids encoding and method for detecting nucleic acid encoding human metabotropic glutamate receptor subtype mGluR6
US6228610B1 (en) 1993-09-20 2001-05-08 Novartis Corporation Human metabotropic glutamate receptor subtypes (hmR4, hmR6, hmR7) and related DNA compounds
EP1199359A1 (fr) * 1993-09-20 2002-04-24 Novartis AG Sous-types de récepteur métabotrope de glutamate humain (HMR4, HMR6, HMR7) et composés d'ADN apparentés
US6515107B2 (en) 1993-09-20 2003-02-04 Novartis Corporation Human metabotropic glutamate receptor 7 subtypes
US7253257B2 (en) 1993-09-20 2007-08-07 Novartis Corporation Human metabotropic glutamate receptor subtype mGluR7b
WO1996029404A1 (fr) * 1995-03-20 1996-09-26 Sibia Neurosciences, Inc. SOUS-TYPE mGluR6 DE RECEPTEUR METABOTROPE DE GLUTAMATE CHEZ L'HOMME
US6384205B1 (en) * 1996-03-12 2002-05-07 Eli Lilly And Company Metabotropic glutamate receptor 4 nucleic acid
WO1999021992A3 (fr) * 1997-10-23 1999-09-10 Ganimed Pharmaceuticals Gmbh Molecules d'acides nucleiques codant pour un recepteur de glutamate
US7262280B1 (en) 1998-04-03 2007-08-28 Nps Pharmaceuticals, Inc. G-protein fusion receptors and constructs encoding same
WO2005069010A1 (fr) * 2004-01-19 2005-07-28 Bayer Healthcare Ag Diagnostics et traitements de pathologiques associees au recepteur metabotropique du glutamate 4 (mglur4) humain
US11254744B2 (en) 2015-08-07 2022-02-22 Imaginab, Inc. Antigen binding constructs to target molecules
US11266745B2 (en) 2017-02-08 2022-03-08 Imaginab, Inc. Extension sequences for diabodies

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