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WO1999060121A1 - PROTEINE DU TYPE RECEPTEUR DE GLUTAMATE METABOTROPIQUE ET ADNc DE CODAGE - Google Patents

PROTEINE DU TYPE RECEPTEUR DE GLUTAMATE METABOTROPIQUE ET ADNc DE CODAGE Download PDF

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Publication number
WO1999060121A1
WO1999060121A1 PCT/IL1999/000265 IL9900265W WO9960121A1 WO 1999060121 A1 WO1999060121 A1 WO 1999060121A1 IL 9900265 W IL9900265 W IL 9900265W WO 9960121 A1 WO9960121 A1 WO 9960121A1
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Prior art keywords
mgrcm
leu
val
ser
ala
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PCT/IL1999/000265
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English (en)
Inventor
Liat Mintz
Kinneret Savitsky
Amir Toporik
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Compugen Ltd.
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Priority to AU39526/99A priority Critical patent/AU3952699A/en
Publication of WO1999060121A1 publication Critical patent/WO1999060121A1/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

Definitions

  • This invention relates to a novel MGIuR-like receptor protein and polynucleotide compositions, to the production of these compositions, and to the use of the compositions in the diagnosis, prevention, and treatment of disease states.
  • Receptors belonging to the 7-transmembrane G-protein coupled receptor (7-TDGR) superfamily are transmembrane proteins present in the plasma membrane characterized by amino acid sequences which contain seven hydrophobic domains predicted to form the membrane-spanning regions. 7-TDGRs transmit extracellular stimuli to the interior of the cell via interactions with G-proteins.
  • the stimuli for different receptors in the superfamily include light, taste, odor, small peptides, amino acid derivatives and lipid analogs (see, e.g., Watson and Arkinstall (1994) The G-Protein Linked Receptor Factsbook, Academic Press, New York).
  • Activation of the 7-TDGR receptor by extracelluar stimuli leads to activation of intracelluar G-proteins, which in turn modulates production of intracellular second messenger molecules such as cAMP and IP 3 .
  • One class of receptors belonging to the 7-TDGR superfamily are the metabotropic glutamate receptors (mGluRs), which are involved in the stimulation of phospholipase C, the presynaptic inhibition of glutamate release, the closure of cation channels in retinal ON bipolar cells, and the modulation of adenylate cyclase.
  • mGluRs metabotropic glutamate receptors
  • eight subtypes have been identified, which share common structural architecture.
  • the mGluRs contain a signal sequence (cleaved during post-translational processing), an extracellular NH -domain, seven transmembrane segments, and a cytosolic C-terminal domain.
  • alternative splicing results in the production of receptor splice variants (Makoff A.J. et al, NeuroReport 8:2943-2947 (1997)).
  • Group I contains mGluRl (and splice variants la, lb, lc, Id, and le) and mGluR5 (and splice variants 5a and
  • Group II contains mGluR2 and mGluR3; and Group III contains mGluR4 (and splice variants 4a and 4b), mGluR ⁇ , mGluR7 (and splice variants 7a and 7b) and mGluR8 (Nicoletti, F. et al, Trends Neurosci. 19:219-224 (1996)).
  • mGluRs within groups show similar (but not identical) biochemical properties.
  • the group I receptors stimulate inositol triphosphate (IP 3 ) production and intracellular Ca mobilization, induce arachadonic acid release, and have nearly identical agonist selectivities (Nakanishi, S., Science 258:597-603 (1992)).
  • the group II and III receptors inhibit the forskolin-stimulated accumulation of intracellular cAMP in an agonist-dependent manner, but with differing agonist selectivities between the two groups.
  • the mGluR2, mGluR3, and mGluR4 receptors are all sensitive to PTX, indicating that the Group II and III receptors are coupled to inhibitory G (Gj) proteins. Furthermore, the various mGluR subtypes show overlapping but non-equivalent expression patterns in mammalian brain. It is notable that receptor subtypes within the same group, though having similar primary sequences, signal transduction properties, and agonist selectivities, are found to be differentially expressed in the CNS (Nakanishi, supra).
  • Glutaminergic neurotransmission is disrupted in many neuropathologic conditions. Imbalances in glutaminergic function have been implicated in neuronal death associated with ischemia, anoxia, stroke, epilepsy, and in neurodegenerative disorders. During brain ischemia and hypoglycemia, neuronal deterioration and cell death results from massive stimulation of glutamate receptors by high concentrations of extracellular glutamate. Glutamate neurotoxicity (or "excitotoxicity”) may also underlie slow-progressing neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, and Parkinson's disease.
  • Receptor proteins and the nucleic acids which encode them have various industrial applications, including as pharmaceutical and diagnostic agents. Receptor proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor-ligand interaction.
  • the discovery of a novel receptor protein having sequence similarity to receptors of the mGluR family, and the polynucleotides which encode it, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of various disease states, such as disorders of the central and peripheral nervous systems, including neurological and neurodegenerative disorders, as well as cardiac, urologic, and gastrointestinal disorders.
  • the invention includes a novel, isolated receptor protein, having sequence similarity to the mGluR class of 7-transmembrane G-protein coupled receptors and identified herein as MGRcm, which comprises a sequence having at least 80 percent sequence identity with SEQ ID NO:2.
  • the protein comprises a sequence at having at least 90% identity to SEQ ID NO:2, or comprises the sequence identified as SEQ ID NO:2.
  • the invention also includes fragments of MGRcm, preferably at least about 10-50 amino acids in length, and as well as pharmaceutical compositions containing MGRcm.
  • the present invention concerns the mouse homolog of MGRcm and concerns a sequence having at least 80% identity, prefarably 90% identity to the sequence identified as SEQ ID NO:4, fragments of said sequences having 10-50 amino acids and pharmaceutical compositions.
  • the invention includes an isolated polynucleotide having a sequence which encodes MGRcm (both human and murine) as described above, or a sequence complementary to the MGRcm coding sequence, and a composition comprising the polynucleotide.
  • the polynucleotide may be mRNA, DNA, cDNA, genomic DNA, as well as an antisense analog thereof.
  • the polynucleotide may encode an MGRcm having at least 80% sequence identity to the protein sequence SEQ ID NO:2 or the sequence of SEQ ID NO: 4.
  • the polynucleotide may contain, for example, a coding sequence having at least 80% sequence identity with the polynucleotide sequence identified as SEQ ID NO:l (coding for SEQ ID NO:2) or the nucleotide sequence identified as SEQ ID NO:3 (coding for SEQ. ID NO:4).
  • the polynucleotide has the sequence identified as SEQ ID NO:l.
  • the composition also contemplates fragments of the polynucleotide, preferably at least about 15-30 nucleotides in length.
  • a recombinant expression vector containing a polynucleotides as described above, and, operably linked to the polynucleotide, regulatory elements effective for expression of the protein in a selected host. Preferred coding sequences are given above.
  • the invention includes a host cell containing the vector.
  • the invention further includes a method for producing MGRcm by recombinant techniques, by cult ⁇ ring recombinant prokaryotic or eukaryotic host cells containing nucleic acid sequences encoding MGRcm under conditions promoting expression of the protein, and subsequent recovery of the protein from the host cell or the cell culture medium.
  • the invention includes an antibody specific against MGRcm.
  • the antibody has diagnostic and therapeutic applications, particularly in treating neurologic and psychiatric disorders and neurodegenerative conditions, such as seizures, epilepsy, amnesia, dementia, migraine, depression, mania, Alzheimer's disease, Huntington's disease, Parkinson's disease, disorders associated with the peripheral nervous system, as well as cardiac, urologic, and gastrointestinal disorders.
  • Treatment methods which employ antisense or coding sequence polynucleotides for inhibiting or enhancing levels of MGRcm are also contemplated, as are treatment methods which employ antibodies specific against MGRcm.
  • a method of detecting a polynucleotide which encodes MGRcm in a biological sample involves the steps of: (a) hybridizing the complement of a polynucleotide which encodes MGRcm to nucleic acid material of a biological sample, thereby forming a hybridization complex, and (b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding MGRcm in the biological sample.
  • Methods for detecting mutations in the coding region of MGRcm are also contemplated.
  • An exemplary method includes (a) contacting a test compound with MGRcm, (b) measuring the effect of the test compound on the activity of the MGRcm, and (c) selecting the test compound as a candidate compound if its effect on the activity of MGRcm is above a selected threshold level.
  • the activity measured may be, for example, MGRcm-mediated production of a second messenger such as cAMP or IP 3 .
  • the test compound is a component of a combinatorial library.
  • the test compound is an antibody specific against MGRcm protein.
  • the invention also includes, in a related aspect, a compound identified by the screening methods described above.
  • Fig. 1A shows the nucleic acid sequence (SEQ ID NO:l) and Fig. IB shows the translated protein sequence (SEQ ID NO :2)of MGRcm;
  • Fig. 2A shows the nucleic acid sequence (SEQ ID NO:3) and Fig. 2B shows the translated protein sequence (SEQ ID NO:4)of a mouse homolog of MGRcm;
  • Fig. 3A shows a pairwise amino acid sequence alignment between MGRcm (SEQ ID NO:2) and human mGluRl (MGR1_HUMAN, GenBank Accession Q 13255);
  • Fig. 3B shows a pairwise amino acid sequence alignment between MGRcm and human mGluRl
  • MGRcm SEQ ID NO:2
  • MGR3_RAT GenBank Accession P31422
  • Fig. 4 shows a hydrophobicity analysis of SEQ ID NO:2, prepared using the TMpred program and the TMbase database.
  • SEQ ID NO: 1 is the nucleic acid sequence of the MGRcm transcript
  • SEQ ID NO:2 is the predicted amino acid translation of MGRcm
  • SEQ ID NO:3 is the nucleic acid sequence of the mouse homolog MGRcm transcript
  • SEQ ID NO:4 is the predicted amino acid translation of transcript of the mouse homolog of MGRcm
  • SEQ ID NO: 5 is the amino acid sequence of human mGluRl (MGR1_HUMAN, GenBank Accession Q13255);
  • SEQ ID NO:6 is the amino acid sequence of rat mGluR3 (MGR3_RAT, GenBank Accession P31422);
  • SEQ ID NO:7 is an oligonucleotide used for northern blot analysis.
  • niGluR-like protein is a protein which has at least 20% amino acid sequence identity to a corresponding aligned region of at least one of the eight known mGluR subtypes.
  • MGRcm refers to an mGluR-like protein which contains a sequence having at least 80 percent, preferably 90 percent, and more preferably 95 percent sequence identity with the polypeptide identified as SEQ ID NO:2.
  • the protein may be a mature MGRcm protein and/or a modified MGRcm protein.
  • reference to MGRcm is meant to include the full-length molecule and fragments thereof unless the context indicates otherwise.
  • the term also refers to the mouse homolog of MGRcm as depicted in SEQ ID NO:4, as well as to sequences having 80% identity, preferably 90% identity, most preferably 95% identity with this sequence.
  • mature MGRcm protein refers to the MGRcm receptor protein as it exists in the cell after post-translational processing, e.g. removal of a signal sequence.
  • modified' ' when referring to a protein of the invention, means a protein which is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art.
  • modifications include, but are not limited to, acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
  • biologically active refers to an MGRcm having structural, regulatory or biochemical functions of the naturally occurring MGRcm, and the ability to modulate intracellular signal transduction, e.g., by the production of intracellular second messengers.
  • immunologically active defines the capability of a natural, recombinant or synthetic MGRcm, or any fragment thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • fragment when referring to MGRcm, means a polypeptide which has an amino acid sequence which is the same as part of but not all of the amino acid sequence of an MGRcm receptor, which either retains essentially the same biological function or activity as MGRcm, or retains at least one of the functions or activities of MGRcm; for example, an extracellular fragment which retains the ability to bind an extracellular ligand of MGRcm, or an intracellular C-terminal fragment which retains signal transduction modulation activity, or a fragment which retains immunological activity of MGRcm.
  • the fragment preferably includes at least 10-50 contiguous residues of MGRcm, more preferably at least 50-200 residues, still more preferably at least 200-500 residues.
  • portion when referring to a protein of the invention, means a polypeptide which has an amino acid sequence which is the same as part of the amino acid sequence of the present invention or a variant thereof, which does not necessarily retain any biological function or activity.
  • a “conservative substitution” refers to the substitution of an amino acid in one class by an amino acid in the same class, where a class is defined by common physicochemical amino acid sidechain properties and high substitution frequencies in homologous proteins found in nature (as determined, e.g., by a standard Dayhoff frequency exchange matrix or BLOSUM matrix).
  • Six general classes of amino acid sidechains include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (He, Leu, Val, Met); and Class VI (Phe, Tyr, Trp).
  • substitution of an Asp for another class III residue such as Asn, Gin, or Glu, is a conservative substitution.
  • non-conservative substitution refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gin.
  • Optimal alignment is defined as an alignment giving the highest percent identity score. Such alignment can be performed using a variety of commercially available sequence analysis programs, such as the local alignment program L ALIGN using a ktup of 1, default parameters and the default PAM. A preferred alignment is the one performed using the CLUSTAL-W program from MacVector, operated with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM similarity matrix. If a gap needs to be inserted into a first sequence to optimally align it with a second sequence, the percent identity is calculated using only the residues that are paired with a corresponding amino acid residue (i.e., the calculation does not consider residues in the second sequences that are in the "gap" of the first sequence).
  • Percent sequence identity refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in two or more optimally aligned polypeptide sequences are identical.
  • a first polypeptide region is said to "correspond" to a second polypeptide region when the regions are essentially co-extensive when the sequences containing the regions are aligned using a sequence alignment program, as above.
  • Corresponding polypeptide regions typically contain a similar, if not identical, number of residues. It will be understood, however, that corresponding regions may contain insertions or deletions of residues with respect to one another, as well as some differences in their sequences.
  • sequence identity means nucleic acid or amino acid sequence identity in two or more aligned sequences, aligned as defined above.
  • Sequence similarity between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • 80% protein sequence similarity means that 80% of the amino acid residues in two or more aligned protein sequences are conserved amino acid residues, i.e. are conservative substitutions.
  • gene means the segment of DNA involved in producing a polypeptide chain; it may include regions preceding and following the coding region, e.g. 5' untranslated (5' UTR) or "leader" sequences and 3'
  • MGRcm is a polynucleotide which contains the coding sequence of MGRcm (i) in isolation, (ii) in combination with additional coding sequences, such as fusion protein or signal peptide, in which the MGRcm coding sequence is the dominant coding sequence, (iii) in combination with non-coding sequences, such as introns and control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host, and/or (iv) in a vector or host environment in which the MGRcm coding sequence is a heterologous gene.
  • heterologous DNA and “heterologous RNA” refer to nucleotides that are not endogenous to the cell or part of the genome in which they are present; generally such nucleotides have been added to the cell, by transfection, micro injection, electroporation, or the like. Such nucleotides generally include at least one coding sequence, but this coding sequence need not be expressed.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • fragment when referring to an MGRcm coding sequence, means a polynucleotide which has a nucleic acid sequence which is the same as part of but not all of the nucleic acid sequence of the MGRcm coding sequence.
  • the polynucleotide fragment preferably includes at least 15 contiguous bases of MGRcm coding sequence, preferably at least 20-30 bases.
  • expression vector refers to vectors that have the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available.
  • substantially purified refers to molecules, either polynucleotides or polypeptides, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
  • a “variant" polynucleotide sequence encodes a "variant" amino acid sequence which is altered by one or more amino acids from the reference polypeptide sequence.
  • the variant polynucleotide sequence may encode a variant amino acid sequence which contains "conservative" substitutions, wherein the substituted amino acid has structural or chemical properties similar to the amino acid which it replaces.
  • the variant polynucleotide sequence may encode a variant amino acid sequence which contains "non-conservative" substitutions, wherein the substituted amino acid has dissimilar structural or chemical properties to the amino acid which it replaces.
  • Variant polynucleotides may also encode variant amino acid sequences which contain amino acid insertions or deletions, or both.
  • An “allelic variant” is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • a “mutant” amino acid or polynucleotide sequence is a variant amino acid sequence, or a variant polynucleotide sequence which encodes a variant amino acid sequence, which has significantly altered biological activity from that of the naturally occurring protein.
  • a “deletion” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
  • insertion or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.
  • substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
  • modulate refers to the change iri activity of the polypeptide of the invention. Modulation may relate to an increase or a decrease in biological activity, binding characteristics, or any other biological, functional, or immunological property of the molecule.
  • agonist refers to a molecule which, when bound to the receptor of the present invention, modulates the activity of the receptor by inducing, increasing, or prolonging the duration of the biological activity mediated by the receptor.
  • Agonists may themselves be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which bind to and modulate the activity of the receptor.
  • Antagonist refers to a molecule which, when bound to the receptor of the present invention, modulates the activity of the receptor by blocking, decreasing, or shortening the duration of the biological activity mediated by the receptor. Antagonists may themselves be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which bind to and modulate the activity of the receptor.
  • humanized antibody refers to antibody molecule in which one or more amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding activity of the antibody.
  • Treating a disease refers to administering a therapeutic substance effective to reduce the symptoms of the disease and/or lessen the severity of the disease.
  • MGRcm is (i) an mGluR-like protein, and (ii) contains an amino acid sequence having at least 80%, preferably 90% or 95%, sequence identity to the amino acid sequence identified as SEQ ID NO:2 or SEQ ID NO:4.
  • Fig. 1 shows a polynucleotide sequence encoding MGRcm, the polynucleotide identified herein as SEQ ID NO:l, and its translation product, SEQ ID NO:2.
  • Fig. 2 shows a polynucleotide sequence encoding a mouse homolog of MGRcm, the polynucleotide identified herein as SEQ ID NO:3, and its translation product, SEQ ID NO:4.
  • Figs. 3A and 3B show pairwise Smith- Waterman amino acid sequence alignments between MGRcm (SEQ ID NO:2)and human mGluRl (MGR1JTUMAN, GenBank Accession Q13255), and between MGRcm (SEQ ID NO:2) and rat mGluR3 (MGR3_RAT, GenBank Accession P31422), respectively. As seen in these figures, MGRcm shares approximately 20% amino acid sequence identity with portions of these mGlu receptor proteins.
  • the invention contemplates mRNA transcripts which encode MGRcm or its mouse homolog, and DNA equivalents of the RNA, including cDNA and chemically synthesized DNA.
  • the polynucleotides of the invention include sequences which encode MGRcm and MGRcm mouse homolog sequences complementary to the protein coding sequence, and novel fragments of the polynucleotide.
  • the polynucleotides may be in the form of RNA or in the form of DNA, and include messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA.
  • the DNA may be double-stranded or single-stranded, and if single-stranded may be the coding strand or the non-coding (anti-sense, complementary) strand.
  • the polynucleotide has at least 70%, preferably at least 80% or 90% sequence identity with the sequence identified as SEQ ID NO:l. In another embodiment, the polynucleotide has at least 70%, preferably at least 80%) or 90% sequence identity with the sequence identified as SEQ ID NO:3.
  • the polynucleotides may include the coding sequence of MGRcm or its mouse homolog (i) in isolation, (ii) in combination with additional coding sequences, such as fusion protein or signal peptide, in which the MGRcm coding sequence is the dominant coding sequence, (iii) in combination with non-coding sequences, such as introns and control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host, and/or (iv) in a vector or host environment in which the MGRcm coding sequence is a heterologous gene.
  • the polynucleotide may encode a soluble fragment of MGRcm (human or murine), which includes the N-terminal extracellular fragment or the C-terminal intracellular fragment of the protein which have been cleaved from the transmembrane domains of the MGRcm receptor.
  • the polynucleotides of the present invention may also have the protein coding sequence fused in-frame to a marker sequence which allows for purification of MGRcm or its mouse homolog.
  • the marker sequence may be, for example, a hexahistidine tag to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al, Ce/737:767 (1984)).
  • polynucleotide fragments also referred to herein as oligonucleotides, typically having at least 15 bases, preferably 20-30 bases corresponding to a region of the coding-sequence polynucleotide.
  • the fragments may be used as probes, primers, antisense agents, and the like, according to known methods.
  • the polynucleotides may be obtained by screening cDNA libraries using oligonucleotide probes which can hybridize to or PCR-amplify polynucleotides which encode the MGRcm receptors and fragments disclosed above.
  • cDNA libraries prepared from a variety of tissues are commercially available and procedures for screening and isolating cDNA clones are well-known to those of skill in the art. Such techniques are described in, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd Edition), Cold Spring Harbor Press, Plainview, N.Y. and Ausubel FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
  • the polynucleotides may be extended to obtain upstream and downstream sequences such as promoters, regulatory elements, and 5' and 3' untranslated regions (UTRs). Extension of the available transcript sequence may be performed by numerous methods known to those of skill in the art, such as PCR or primer extension (Sambrook et al., supra), or by the RACE method using, for example, the Marathon RACE kit (Clontech, Cat. # K1802-1).
  • genomic DNA is amplified in the presence of primer to a linker sequence and a primer specific to the known region.
  • the amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR can be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al, Nucleic Acids Res. 16:8186, (1988)).
  • the primers may be designed using OLIGO(R) 4.06 Primer Analysis Software (1992); National Biosciences Inc, Madison, Minn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Capture PCR (Lagerstrom M et al (1991) PCR Methods Applic 1 : 111-19) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into a flanking part of the DNA molecule before PCR.
  • flanking sequences Another method which may be used to retrieve flanking sequences is that of Parker, J.D., et al., Nucleic Acids Res. 19:3055-60, (1991)). Additionally, one can use PCR, nested primers and PromoterFinderTM libraries to "walk in" genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • Preferred libraries for screening for full length cDNAs are ones that have been size-selected to include larger cDNAs.
  • random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes. A randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA. Genomic libraries are useful for extension into the 5' nontranslated regulatory region.
  • polynucleotides and oligonucleotides of the invention can also be prepared by solid-phase methods, according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized, then joined to form continuous sequences up to several hundred bases.
  • polynucleotide coding sequences and novel oligonucleotides of the invention have a variety of uses in (1) synthesis of MGRcm, (2) diagnostics, (3) gene mapping, and (4) therapeutics.
  • polynucleotide sequences which encode MGRcm, its mouse homolog, fragments of the protein, fusion proteins, or functional equivalents thereof may be used in recombinant DNA molecules that direct the expression of MGRcm in appropriate host cells. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used to clone and express MGRcm.
  • Codons preferred by a particular prokaryotic or eukaryotic host can be selected, for example, to increase the rate of MGRcm polypeptide expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
  • polynucleotide sequences of the present invention can be engineered in order to alter an MGRcm coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product.
  • alterations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to produce splice variants, etc.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook, et al, (supra).
  • the present invention also relates to host cells which are genetically engineered with vectors of the invention, and the production of proteins and polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (i.e., transduced, transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the MGRcm gene.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art.
  • the polynucleotides of the present invention may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and related sub-cloning procedures are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis.
  • promoters include: LTR or SV40 promoter, the E. coli lac or trp promoter, the phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation, and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as described above, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • appropriate expression hosts include: bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9; mammalian cells such as CHO, COS, BHK, HEK 293 or Bowes melanoma; adenoviruses; plant cells, etc. It is understood that not all cell lines will be capable of functional coupling of the receptor to the cell's second messenger systems.
  • an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the invention is not limited by the host cells employed.
  • a number of expression vectors may be selected depending upon the use intended for MGRcm. For example, when large quantities of MGRcm are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable. Such vectors include, but are not limited to, multifunctional E.
  • coli cloning and expression vectors such as Bluescript(R) (Stratagene), in which the MGRcm coding sequence may be ligated into the vector in- frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster J. Biol Chem. 264:5503-5509, (1989)); pET vectors (Novagen, Madison WI); and the like.
  • Bluescript(R) Stratagene
  • pIN vectors Van Heeke & Schuster J. Biol Chem. 264:5503-5509, (1989)
  • pET vectors Novagen, Madison WI
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.
  • viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al, Nature 310:511-514, (1984)) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al. EMBO J. 3:17-311, (1987)).
  • plant promoters such as the small subunit of RUBISCO (Coruzzi et al, EMBO J. 3: 1671-1680, (1984); Brogue et al, Science 224:838-843, (9184)); or heat shock promoters (Winter J and Sinibaldi RM, Results. Probl Cell Differ. 17:85-105, (1991)) may be used.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
  • pathogen-mediated transfection see McGraw Hill Yearbook of Science and Technology, McGraw Hill, New York, N.Y., pp 191-196; or Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, New York, N.Y., pp 421-463.
  • MGRcm may also be expressed in an insect system.
  • Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda Sf9 cells or in Trichoplusia larvae.
  • the MGRcm coding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of MGRcm coding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S.
  • an MGRcm coding sequence may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing MGRcm in infected host cells (Logan and Shenk, Proc. Natl Acad. Sci. 81:3655-59, (1984)).
  • transcription enhancers such as the rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV rous sarcoma virus
  • Specific initiation signals may also be required for efficient translation of an MGRcm coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where MGRcm coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic.
  • Enhancers appropriate to the cell system in use (Scharf D et al, Results Probl Cell Differ. 20:125-62, (1994); Bittner et al, Methods in Enzymol, 153:516-544, (1987)).
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., and Battey, I., Basic Methods in Molecular Biology, (1986)).
  • Cell-free translation systems can also be employed to produce polypeptides using RNAs derived from the DNA constructs of the present invention.
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, BHK, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
  • cell lines which stably express MGRcm may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M et al, Cell 11:223-32, (1977)) and adenine phosphoribosyltransferase (Lowy I et al, Cell 22:817-23, (1980)) genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler M et al. Proc. Natl Acad. Sci.
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin F. et al, J. Mol Biol. 150:1-14, (1981)) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman SC and RC Mulligan, Proc. Natl. Acad. Sci.
  • Host cells transformed with a nucleotide sequence encoding MGRcm may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides encoding MGRcm can be designed with signal sequences which direct secretion of MGRcm polypeptide through a prokaryotic or eukaryotic cell membrane.
  • MGRcm may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, Wash.).
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp, Seattle, Wash.
  • the inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and MGRcm is useful to facilitate purification.
  • One such expression vector provides for expression of a fusion protein compromising MGRcm (e.g., a soluble MGRcm fragment) fused to a polyhistidine region separated by
  • the histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath et al, Protein Expression and Purification 3:263-281, (1992)) while the enterokinase cleavage site provides a means for isolating MGRcm from the fusion protein.
  • pGEX vectors Promega, Madison, Wis.
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.
  • ligand-agarose beads e.g., glutathione-agarose in the case of GST-fusions
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art.
  • MGRcm can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. D2. Diagnostic applications
  • the polynucleotides of the present invention may be used for a variety of diagnostic purposes.
  • the polynucleotides may be used to detect and quantitate expression of MGRcm or its splice variants in patient's cells, e.g. biopsied tissues, by detecting the presence of mRNA coding for MGRcm receptor or its splice variants.
  • This assay typically involves obtaining total mRNA from the tissue and contacting the mRNA with a nucleic acid probe.
  • the probe is a nucleic acid molecule of at least 15 nucleotides, preferably 20-30 nucleotides, capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding MGRcm under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of MGRcm.
  • This assay can be used to distinguish between absence, presence, and excess expression of MGRcm and to monitor levels of MGRcm expression during therapeutic intervention.
  • This assay can also be used to identify the presence of alternatively spliced transcripts of MGRcm, by designing multiple probes corresponding to different exons, and/or probes which preferentially hybridize to particular exon/intron interfaces.
  • the invention also contemplates the use of the polynucleotides as a diagnostic for diseases resulting from inherited defective MGRcm genes. These genes can be detected by comparing the sequences of the defective (i.e., mutant) MGRcm gene with that of a normal one. Association of a mutant MGRcm gene with abnormal MGRcm activity may be verified.
  • mutant MGRcm genes can be inserted into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, or complementation experiments in an MGRcm-deficient strain of mammalian cells, e.g. non-neuronal cells such as CHO or BHK) as yet another means to verify or identify mutations.
  • a functional assay system e.g., colorimetric assay, or complementation experiments in an MGRcm-deficient strain of mammalian cells, e.g. non-neuronal cells such as CHO or BHK
  • mutant genes have been identified, one can then screen populations of interest for carriers of the mutant gene.
  • Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limited to such as from blood, urine, saliva, placenta, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki, et al, Nature 324:163-166, (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose.
  • PCR primers complementary to the nucleic acid of the present invention can be used to identify and analyze mutations in the gene of the present invention. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA sequences of the invention. Sequence changes at specific locations may also be revealed by nuclease protection assays, such RNase and SI protection or the chemical cleavage method (e.g. Cotton, et al, Proc. Natl. Acad. Sci. USA 85:4397-4401, (1985)), or by differences in melting temperatures. "Molecular beacons" (Kostrikis L.G.
  • hairpin-shaped, single-stranded synthetic oligonucleotides containing probe sequences which are complementary to the nucleic acid of the present invention may also be used to detect point mutations or other sequence changes as well as monitor expression levels of MGRcm. Such diagnostics would be particularly useful for prenatal testing.
  • Another method for detecting mutations uses two DNA probes which are designed to hybridize to adjacent regions of a target, with abutting bases, where the region of known or suspected mutation(s) is at or near the abutting bases.
  • the two probes may be joined at the abutting bases, e.g., in the presence of a ligase enzyme, but only if both probes are correctly base paired in the region of probe junction.
  • the presence or absence of mutations is then detectable by the presence or absence of ligated probe.
  • oligonucleotide array methods based on sequencing by hybridization (SBH), as described, for example, in U.S. Patent No. 5,547,839.
  • SBH sequencing by hybridization
  • the DNA target analyte is hybridized with an array of oligonucleotides formed on a microchip.
  • the sequence of the target can then be "read” from the pattern of target binding to the array.
  • D3. Gene mapping
  • the sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome.
  • chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the MGRcm cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, which would complicate the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • Polynucleotides which encode MGRcm, or complements of the polynucleotides, may also be used for therapeutic purposes. Expression of MGRcm may be modulated through antisense technology, which controls gene expression through complementary polynucleotides, i.e. antisense DNA or RNA, to the control, 5' or regulatory regions of the gene encoding MGRcm.
  • antisense technology which controls gene expression through complementary polynucleotides, i.e. antisense DNA or RNA, to the control, 5' or regulatory regions of the gene encoding MGRcm.
  • the 5' coding portion of the polynucleotide sequence which codes for the protein of the present invention is used to design an antisense oligonucleotide of from about 10 to 40 base pairs in length. Oligonucleotides derived from the transcription start site, e.g. between positions -10 and +10 from the start site, are preferred.
  • An antisense DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (Lee et al., Nucl Acids Res. 3:173, (1979); Cooney et al, Science 241:456, (1988); and Dervan et al, Science 251:1360, (1991)), thereby preventing transcription and the production of MGRcm.
  • An antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into MGRcm protein (Okano, J. Neurochem., 56:560, (1991)).
  • the antisense constructs can be delivered to cells by procedures known in the art such that the antisense RNA or DNA may be expressed in vivo.
  • compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • polypeptides, and agonist and antagonist compounds which are polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy.
  • Cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • DNA or RNA polynucleotide
  • Such methods are well-known in the art.
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • Retroviruses from which the retroviral plasmid vectors mentioned above may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, psi-2, psi-AM, PA12, T19-14X, VT-19-17-H2, psi-CRE, psi-CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1:5-14, (1990).
  • the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides.
  • retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
  • the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • the genes introduced into cells may be placed under the control of inducible promoters, such as the radiation-inducible Egr-1 promoter, (Maceri, H.J., et al, Cancer Res, 56(19):4311 (1996)), to stimulate MGRcm production or antisense inhibition in response to radiation, e.g., radiation therapy for treating tumors.
  • inducible promoters such as the radiation-inducible Egr-1 promoter, (Maceri, H.J., et al, Cancer Res, 56(19):4311 (1996)
  • MGRcm Receptor Hydrophobicity analysis shows that the 565-residue MGRcm sequence (SEQ ID NO:2) contains up to 8 potential membrane-spanning domains in the N-terminal region of the protein.
  • MGRcm possesses the characteristic architecture of a 7-transmembrane (7tm) receptor, with a putative signal peptide, a relatively short extracellular domain extending from about residue 20 to about residue 40, a group of 7 transmembrane segments extending from about residue 40 to about residue 300, and a relatively long C-terminal cytosolic domain extending from about residue 300 to residue 565.
  • FIGS 3A and 3B show that MGRcm shares approximately 20% amino acid sequence identity with portions of human mGluRl (MGR1_F1UMAN, GenBank Accession Q 13255) and rat mGluR3 (MGR3_RAT, GenBank Accession P31422). Furthermore, using the program Profilesearch, the region corresponding to residues 92-262 of SEQ ID NO:2 attained a highly significant normalized score (N score) of 10.7651 against the profile PS50259 (G_PROTEIN_RECEPTOR_F3_4; PROSITE database). This score is above the N score cutoff of 8.5 for this profile, providing further evidence that MGRcm is a novel member of the metabotropic glutamate / GABA receptor class of 7tm G-protein coupled receptors.
  • N score highly significant normalized score
  • the substantially purified MGRcm of the invention includes a protein containing an amino acid sequence having at least 80%, preferably at least 90% or 95% identity to the sequence identified as SEQ ID NO:2 or SEQ ID NO:4.
  • the protein may be a recombinant protein, a natural protein or a synthetic protein, preferably a recombinant protein.
  • the protein may be in mature and/or modified form, also as defined above.
  • protein fragments having at least 10-50 contiguous amino acid residues derived from MGRcm are also contemplated.
  • sequence variations are preferably those that are considered conserved substitutions, as defined above.
  • a protein with a sequence having at least 80% sequence identity with the protein identified as SEQ ID NO:2 (565 amino acid residues) contains up to 113 amino acid substitutions, preferably conserved substitutions as defined above.
  • the protein has or contains the sequence identified as SEQ ID NO:2 or SEQ ID NO:4.
  • MGRcm may be (i) a protein in which one or more of the amino acid residues in a sequence listed above are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), or (ii) a protein in which one or more of the amino acid residues includes a substituent group, or (iii) a protein in which the MGRcm is fused with another compound, such as a compound to increase the half-life of the protein (for example, polyethylene glycol (PEG)), or (iv) a protein in which additional amino acids are fused to MGRcm, or (v) an isolated fragment of the protein which is soluble, i.e. not membrane bound, yet still binds its natural ligands. Such fragments, variants and derivatives are deemed to be within the scope of those skilled in the art from the teachings herein.
  • fragments and portions of MGRcm may be produced by direct peptide synthesis using solid-phase techniques (cf Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield, J., J. Am. Chem. Soc, 85:2149-2154, (1963)).
  • In vitro peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer.
  • Portions of MGRcm may be chemically synthesized separately and combined using chemical methods.
  • the receptor protein may also be obtained by isolation from natural sources, e.g., by affinity purification using the anti-MGRcm antibody described in the section below (either from humans or from mice cells).
  • the MGRcm receptor of the invention has uses in (1) therapeutic treatment methods and (2) drug screening.
  • MGRcm proteins of the present invention are generally useful in treating diseases and disorders which respond to the modulation of signal transduction via the central and/or peripheral nervous system.
  • diseases and disorders which respond to the modulation of signal transduction via the central and/or peripheral nervous system.
  • cardiac, urologic, and gastrointestinal disorders include cardiac, urologic, and gastrointestinal disorders, neurodegenerative disorders, and neuronal degradation associated with, for example, ischemia, anoxia, stroke, and epilepsy, and other disorders relating to overstimulation of the sympathetic nervous system including hypoglycemia, vasoconstriction, renal failure, arrhythmia, peripheral vascular disorders, heart failure, nephrosis, cirrhosis, dysphagia, and gastritis.
  • a polypeptide fragment of MGRcm may be employed to inhibit activity of MGRcm by binding an agonist which is essential for MGRcm activity and thus preventing the agonist from interacting with MGRcm.
  • a fragment of MGRcm preferably a soluble fragment, may alternatively be employed to block the binding of agonists to MGRcm, thus likewise preventing the cellular response induced by the binding of the agonist to the MGRcm receptor.
  • MGRcm compositions are tested in appropriate in vitro and in vivo animal models of disease, to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art.
  • MGRcm compositions may be administered by any of a number of routes and methods designed to provide a consistent and predictable concentration of compound at the target organ or tissue.
  • the polypeptide compositions may be administered alone or in combination with other agents, such as stabilizing compounds, and/or in combination with other pharmaceutical agents such as drugs or hormones.
  • MGRcm compositions may be administered by a number of routes including, but not limited to oral, intravenous, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means. MGRcm compositions may also be administered via liposomes. Such administration routes and appropriate formulations are generally known to those of skill in the art.
  • the polypeptide can be given via intravenous or intraperitoneal injection. Similarly, the polypeptide may be injected to other localized regions of the body.
  • the polypeptide may also be administered via nasal insufflation. Enteral administration is also possible.
  • the polypeptide should be formulated into an appropriate capsule or elixir for oral administration, or into a suppository for rectal administration.
  • the foregoing exemplary administration modes will likely require that the polypeptides be formulated into an appropriate carrier, including ointments, gels, suppositories. Appropriate formulations are well known to persons skilled in the art. Dosage of the polypeptide will vary, depending upon the potency and therapeutic index of the particular polypeptide selected. These parameters are easily determinable by the skilled practitioner.
  • the polypeptide inhibits neuronal cell degradation in vitro at a given concentration
  • the practitioner will know that the final desired therapeutic concentration will be this range, calculated on the basis of expected biodistribution.
  • An appropriate target concentration is in the ng/kg to low mg/kg range, e.g., 50 ng/kg to 1 mg/kg body weight, for IV administration.
  • a therapeutic composition for use in the treatment method can include the polypeptide in a sterile injectable solution, the polypeptide in an oral delivery vehicle, or the polypeptide in a nebulized form, all prepared according to well known methods.
  • Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the present invention also includes an assay for identifying molecules, such as synthetic drugs, antibodies, peptides, or other molecules, which have a modulating effect on the activity of MGRcm, e.g. agonists or antagonists of the MGRcm receptor of the present invention.
  • an assay comprises the steps of providing a functional MGRcm receptor encoded by the polynucleotides of the present invention, contacting the MGRcm receptor with one or more molecules to determine its modulating effect on the activity of the receptor, and selecting from the molecules a candidate molecule capable of modulating MGRcm receptor activity.
  • Such compounds are useful in the treatment of disease conditions associated with activation or depression of MGRcm activity.
  • MGRcm its catalytic or immunogenic fragments or oligopeptides thereof, can be used for screening therapeutic compounds in any of a variety of drug screening techniques.
  • the protein employed in such a test may be membrane-bound, free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • the formation of binding complexes between MGRcm and the agent being tested may be measured.
  • Compounds which inhibit binding between MGRcm and its agonists may also be measured.
  • the screening system includes recombinantly expressed MGRcm, and the compounds screened are tested for their ability to block or enhance the signal transduction activity of MGRcm.
  • mammalian cell lines which lack MGRcm receptor are used to express MGRcm which interacts with G protein(s) present in the cell line.
  • compounds are screened for their relative affinity as receptor agonists or antagonists by comparing the relative receptor occupancy to the extent of ligand-induced stimulation or inhibition of second messenger production, such as cAMP or IP 3 .
  • Kits for quantitating the production of second messenger molecules are commercially available (e.g., Amersham)
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the MGRcm protein is described in detail by Geysen in PCT Application WO 84/03564, published on Sep. 13, 1984.
  • peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with soluble fragments of MGRcm, or intact MGRcm solubilized in detergents or in lipid vesicles, and washed. Bound MGRcm is then detected by methods well known in the art.
  • Substantially purified MGRcm can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • Antibodies to the MGRcm may also be used in screening assays according to methods well known in the art. For example, a "sandwich" assay may be performed, in which an anti-MGRcm antibody is affixed to a solid surface such as a microtiter plate and solubilized MGRcm is added. Such an assay can be used to capture compounds which bind to MGRcm. Alternatively, such an assay may be used to measure the ability of compounds to interfere with the binding of an MGRcm agonist to MGRcm.
  • purified MGRcm is used to produce anti-MGRcm antibodies which have diagnostic and therapeutic uses related to the activity, distribution, and expression of MGRcm.
  • Antibodies to MGRcm may be generated by methods well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library. Antibodies, i.e., those which block ligand binding, are especially preferred for therapeutic use.
  • MGRcm for antibody induction does not require biological activity; however, the protein fragment or oligopeptide must be antigenic.
  • Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids. Preferably they should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of a MGRcm polypeptide may be fused with another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to MGRcm. For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with MGRcm protein or any portion, fragment or oligopeptide which retains immunogenic properties.
  • adjuvants may be used to increase immunological response.
  • adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are potentially useful human adjuvants.
  • Monoclonal antibodies to MGRcm may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al, Proc. Natl Acad. Sci, 86:3833-3837, (1989)), and Winter, G. and Milstein C, Nature, 349:293-299, (1991)).
  • Antibody fragments which contain specific binding sites for MGRcm may also be generated.
  • such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W.D., et al, Science 256:1275-1281, (1989)).
  • a variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the formation of complexes between MGRcm and its specific antibody and the measurement of complex formation.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on MGRcm is preferred, but a competitive binding assay may also be employed. These assays are described in Maddox D.E. et al, J. Exp. Med. 158:1211, (1983).
  • Antibodies which specifically bind MGRcm protein are useful for the diagnosis of conditions or diseases characterized by expression of MGRcm.
  • Such antibodies may be used in assays to monitor patients being treated with MGRcm, its agonists, or its antagonists.
  • Diagnostic assays for MGRcm protein include methods utilizing the antibody and a label to detect MGRcm in extracts of cells or tissues.
  • the proteins and antibodies of the present invention may be used with or without modification. Frequently, the proteins and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule. A wide variety of reporter molecules are known in the art.
  • a variety of protocols for measuring MGRcm, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS). As noted above, a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on MGRcm is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, et al. (supra). Such protocols provide a basis for diagnosing altered or abnormal levels of MGRcm expression.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • Normal or standard values for MGRcm expression are established by combining cell extracts taken from normal subjects, preferably human, with antibody to MGRcm under conditions suitable for complex formation which are well known in the art.
  • the amount of standard complex formation may be quantified by various methods, preferably by photometric methods.
  • standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by disease. Deviation between standard and subject values establishes the presence of disease state.
  • the antibody assays are useful to determine the level of MGRcm present in a particular tissue, e.g., biopsied tumor tissue or neuronal tissue, as an indication of whether MGRcm is being overexpressed or underexpressed in the tissue, or as an indication of how MGRcm levels are responding to drug treatment.
  • therapeutic value may be achieved by administering an antibody specific against MGRcm to inhibit the action of MGRcm by, e.g., inhibiting the binding of agonists to the MGRcm receptor, to treat conditions generally associated with overstimulation of the mGluR class of receptors.
  • Such conditions include those generally classed as cognitive disorders, including dementia, delirium, and amnesic disorders.
  • MGRcm antibody therapy includes neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, and Parkinson's disease; neuronal cell death associated with, for example, ischemia, anoxia, stroke, and epilepsy; and other disorders relating to overstimulation of the sympathetic nervous system including hypoglycemia, vasoconstriction, renal failure, arrhythmia, peripheral vascular disorders, heart failure, nephrosis, cirrhosis, dysphagia, and gastritis.
  • neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, and Parkinson's disease
  • neuronal cell death associated with, for example, ischemia, anoxia, stroke, and epilepsy
  • disorders relating to overstimulation of the sympathetic nervous system including hypoglycemia, vasoconstriction, renal failure, arrhythmia, peripheral vascular disorders, heart failure, nephrosis, cirrhosis, dysphagia, and gastritis.
  • the antibody employed is preferably a humanized monoclonal antibody, or a human Mab produced by known globulin-gene library methods.
  • the antibody is administered typically as a sterile solution by IV injection, although other parenteral routes may be suitable.
  • the antibody is administered in an amount between about 1-15 mg/kg body weight of the subject. Treatment is continued, e.g., with dosing every 1-7 days, until a therapeutic improvement is seen.
  • the antibody treatment is used to treat neuronal cell death associated with sudden-onset conditions such as ischemia, anoxia, stroke, or epilepsy, by administering the antibody with the first observable signs of the episode.
  • the antibody may also be used to treat epileptics or those prone to stroke, seizures, or hypoglycemia, by administering the antibody prior to the episodes.
  • Treatment for chronic disorders such as long-term neurodegenerative conditions is also contemplated, in this case, with long term injection of antibody.

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Abstract

L'invention concerne une protéine-récepteur du type MgluR (MGRcm) et des polynucléotides identifiant et codant la protéine MGRcm en question. L'invention concerne également des vecteurs d'expression et des cellules hôtes renfermant une séquence d'acides nucléiques codant la protéine MGRcm. L'invention concerne en outre des procédés de diagnostic et de traitement relatifs aux maladies associées à l'expression de la protéine MGRcm, des analyses de criblage faisant appel à ladite protéine, un nucléotide et des compositions d'anticorps.
PCT/IL1999/000265 1998-05-19 1999-05-19 PROTEINE DU TYPE RECEPTEUR DE GLUTAMATE METABOTROPIQUE ET ADNc DE CODAGE WO1999060121A1 (fr)

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AU39526/99A AU3952699A (en) 1998-05-19 1999-05-19 Metabotropic glutamate receptor-like protein and encoding cdna

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000015793A3 (fr) * 1998-09-17 2000-09-28 Incyte Pharma Inc Proteines gpcr humaines

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0679716A1 (fr) * 1993-11-12 1995-11-02 Kenichi Matsubara Signature genique
WO1996006167A1 (fr) * 1994-08-19 1996-02-29 Novartis Ag Recepteur de glutamate
WO1999006550A2 (fr) * 1997-08-01 1999-02-11 Genset Marqueurs sequences 5' exprimes pour proteines secretees exprimees dans la prostate
WO1999031117A1 (fr) * 1997-12-18 1999-06-24 Human Genome Sciences, Inc. 110 proteines secretees humaines

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0679716A1 (fr) * 1993-11-12 1995-11-02 Kenichi Matsubara Signature genique
WO1996006167A1 (fr) * 1994-08-19 1996-02-29 Novartis Ag Recepteur de glutamate
WO1999006550A2 (fr) * 1997-08-01 1999-02-11 Genset Marqueurs sequences 5' exprimes pour proteines secretees exprimees dans la prostate
WO1999031117A1 (fr) * 1997-12-18 1999-06-24 Human Genome Sciences, Inc. 110 proteines secretees humaines

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Title
KNÖPFEL T. AND GASPARINI F.: "Metabotropic Glutamate Receptors: potential drug targets", DRUG DISCOVERY TODAY, vol. 1, 1 January 1996 (1996-01-01), pages 103 - 108, XP000650306, ISSN: 1359-6446 *
KNÖPFEL T. ET AL.: "Metabotropic Glutamate Receptors: novel targets for drug development", JOURNAL OF MEDICINAL CHEMISTRY, vol. 38, no. 9, 28 April 1995 (1995-04-28), pages 1417 - 1426, XP000611483, ISSN: 0022-2623 *
PIN J.-P. AND DUVOISIN R.: "Neurotransmitter receptors I. The metabotropic glutamate receptors: structure and functions", NEUROPHARMACOLOGY, vol. 34, no. 1, 1995, pages 1 - 26, XP002113327 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000015793A3 (fr) * 1998-09-17 2000-09-28 Incyte Pharma Inc Proteines gpcr humaines

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