CA2329153A1 - Novel g-protein coupled receptor - Google Patents
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Abstract
The present invention relates to a novel human DNA
sequence encoding a G-protein coupled receptor having homology to the endothelin receptor, the protein encoded by the DNA, and uses thereof.
sequence encoding a G-protein coupled receptor having homology to the endothelin receptor, the protein encoded by the DNA, and uses thereof.
Description
TITLE OF TIC INVENTION
NOVEL G-PROTEIN COUPLED RECEPTOR
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention is directed to a novel human DNA sequence encoding a G-protein coupled receptor having homology to the endothelin receptor, the protein encoded by the DNA, and uses thereof.
BACKGROUND OF THE INVENTION
G-protein coupled receptors (GPCRs) are a very large class of membrane receptors that relay information from the exterior to the interior of cells.
GPCRs function by interacting with a class of heterotrimeric proteins known as G-proteins. Most GPCRs function by a similar mechanism. Upon the binding of agonist, a GPCR catalyzes the dissociation of guanosine diphosphate (GDP) from the a subunit of G proteins. This allows for the binding of guanosine triphosphate (GTP) to the a subunit, resulting in the disassociation of the a subunit from the (3 and y subunits. The freed a subunit then interacts with other cellular ocmpenents, and in the process passes on the extracellular signal. represented by the presence of the agonist.
Occasionally, it is the freed ~i and r subunits which transduce the agonist signal.
GPCRs possess common structural characteristics. They have seven hydrophobic domains, about 20-30 amino acids long, linked by sequences of hydrophilic amino acids of varied length. These seven hydrophobic domains intercalate into the plasma membrane, giving rise to a protein with seven transmembrane domains, an extracellular amino terminus, and an intracellular carboxy terminus (Strader et al., 1994, Ann. Rev. Biochem. 63:101-132; S,chertler et al., 1993, Nature 362:770-7721; Dohlman et al., 1991, Ann. Rev. Biochem. 60:653-688).
GPCRs are expressed in a wide variety of tissue types and respond to a wide range of ligands, e.g., protein hormones, biogenic amines, peptides, lipid derived messengers, etc. Given their wide range of expression and ligands, it is not surprising that GPCRs are involved in many pathological states. This has led to great interest in developing modulators of GPCR activity that can be used pharmacologically. For example, Table 1 of Stadel et al., 1997, Trends Pharmacol. Sci. 18:430-437, lists 37 different marketed drugs that act upon GPCRs. Accordingly, there is a great need to understand GPCR function and to develop agents that can be used to modulate GPCR
activity.
SUMMARY OF THE INVENTION
The present invention is directed to a novel human DNA that encodes a G-protein coupled receptor, HGO 1. The DNA encoding HGO1 is substantially free from other nucleic acids and has the nucleotide sequence shown in SEQ.117.N0.:1.
Also provided is an HGO1 protein encoded by the novel DNA sequence. The HG01 protein is substantially free from other proteins and has the amino acid sequence shown in SEQ.D~.N0.:2. Methods of expressing HGO1 in recombinant systems and of identifying agonists and antagonists of HGO l are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 A-B shows the complete cDNA sequence of HG01 (SEQ.117.N0.:1).
Figure 2 shows the complete amino acid sequence of HGO 1 (SEQ.ID.N0.:2). Indicated are the locations of the signal peptide, the mature peptide, and the seven transmembrane domains.
Figure 3 shows the results of a Northern blot of the expression of HGO1 mRNA in various tissues.
Figure 4 shows the alignment of the HGO1 amino acid sequence with the amino acid sequences of the Eta and Etb endothelia receptors.
NOVEL G-PROTEIN COUPLED RECEPTOR
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention is directed to a novel human DNA sequence encoding a G-protein coupled receptor having homology to the endothelin receptor, the protein encoded by the DNA, and uses thereof.
BACKGROUND OF THE INVENTION
G-protein coupled receptors (GPCRs) are a very large class of membrane receptors that relay information from the exterior to the interior of cells.
GPCRs function by interacting with a class of heterotrimeric proteins known as G-proteins. Most GPCRs function by a similar mechanism. Upon the binding of agonist, a GPCR catalyzes the dissociation of guanosine diphosphate (GDP) from the a subunit of G proteins. This allows for the binding of guanosine triphosphate (GTP) to the a subunit, resulting in the disassociation of the a subunit from the (3 and y subunits. The freed a subunit then interacts with other cellular ocmpenents, and in the process passes on the extracellular signal. represented by the presence of the agonist.
Occasionally, it is the freed ~i and r subunits which transduce the agonist signal.
GPCRs possess common structural characteristics. They have seven hydrophobic domains, about 20-30 amino acids long, linked by sequences of hydrophilic amino acids of varied length. These seven hydrophobic domains intercalate into the plasma membrane, giving rise to a protein with seven transmembrane domains, an extracellular amino terminus, and an intracellular carboxy terminus (Strader et al., 1994, Ann. Rev. Biochem. 63:101-132; S,chertler et al., 1993, Nature 362:770-7721; Dohlman et al., 1991, Ann. Rev. Biochem. 60:653-688).
GPCRs are expressed in a wide variety of tissue types and respond to a wide range of ligands, e.g., protein hormones, biogenic amines, peptides, lipid derived messengers, etc. Given their wide range of expression and ligands, it is not surprising that GPCRs are involved in many pathological states. This has led to great interest in developing modulators of GPCR activity that can be used pharmacologically. For example, Table 1 of Stadel et al., 1997, Trends Pharmacol. Sci. 18:430-437, lists 37 different marketed drugs that act upon GPCRs. Accordingly, there is a great need to understand GPCR function and to develop agents that can be used to modulate GPCR
activity.
SUMMARY OF THE INVENTION
The present invention is directed to a novel human DNA that encodes a G-protein coupled receptor, HGO 1. The DNA encoding HGO1 is substantially free from other nucleic acids and has the nucleotide sequence shown in SEQ.117.N0.:1.
Also provided is an HGO1 protein encoded by the novel DNA sequence. The HG01 protein is substantially free from other proteins and has the amino acid sequence shown in SEQ.D~.N0.:2. Methods of expressing HGO1 in recombinant systems and of identifying agonists and antagonists of HGO l are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 A-B shows the complete cDNA sequence of HG01 (SEQ.117.N0.:1).
Figure 2 shows the complete amino acid sequence of HGO 1 (SEQ.ID.N0.:2). Indicated are the locations of the signal peptide, the mature peptide, and the seven transmembrane domains.
Figure 3 shows the results of a Northern blot of the expression of HGO1 mRNA in various tissues.
Figure 4 shows the alignment of the HGO1 amino acid sequence with the amino acid sequences of the Eta and Etb endothelia receptors.
DETAILED DESCRIPTION OF THE INVENTION
For the purposes of this invention;
"Substantially free from other proteins" means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other proteins.
Thus, an HGO1 protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-HGO 1 proteins. Whether a given HGO1 protein preparation is substantially free from other proteins can be determined by such conventional techniques of assessing protein purity as, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting.
"Substantially free from other nucleic acids" means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other nucleic acids. Thus, an HGO1 DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-HGO1 nucleic acids. Whether a given HGO1 DNA preparation is substantially free from other nucleic acids can be determined by such conventional techniques of assessing nucleic acid purity as, e.g., agarose gel electrophoresis combined with appropriate staining methods, e.g., ethidium bromide staining, or by sequencing.
A polypeptide has "substantially the same biological activity" as HGO1 if that polypeptide has a Kd for a ligand that is no more than 5-fold greater than the Kd of HGO 1 for the same ligand.
A "conservative amino acid substitution" refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue.
Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, vaiine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid).
The present invention relates to the identification and cloning ofHG0l, a novel G-protein coupled receptor. The present invention provides DNA
encoding HGO1 that is substantially free from other nucleic acids. The present invention also provides recombinant DNA molecules encoding HGO1.
The present invention provides a DNA molecule substantially free from other nucleic acids having the nucleotide sequence shown in Figure 1 as SEQ.ID.NO.:1. Analysis of SEQ.ID.NO.:1 revealed that it contains an open reading frame at positions 84-1,526. Thus, the present invention also provides a DNA
molecule substantially free from other nucleic acids comprising the nucleotide sequence of positions 84-1,526 of SEQ.117.N0.:1. The present invention also provides recombinant DNA molecules comprising the nucleotide sequence of positions 84-1,526 of SEQ.m.N0.:1.
Sequence analysis of the open reading frame of the HGO1 DNA
revealed that it encodes a protein of 481 amino acids with a predicted signal peptide.
Based on its predicted amino acid sequence, HGO1 most likely represents a novel GPCR. Northern blot analysis showed that HGO1 RNA is highly expressed in the brain and not in the pancreas, kidney, skeletal muscle, liver, lung, placenta, or heart.
Based upon amino acid sequence alignments, HGO1 appears to be related to the endothelin receptors (Figure 4).
The novel DNA sequences of the present invention encoding HGO l, in whole or in part, can be linked with other DNA sequences, i. e., DNA sequences to which HGO1 is not naturally linked, to form "recombinant DNA molecules"
containing HGO1. Such other sequences can include DNA sequences that control transcription or translation such as, e.g., translation initiation sequences, promoters for RNA
polymerase II, transcription or translation termination sequences, enhancer sequences, sequences that control replication in microorganisms, or that confer antibiotic resistance. The novel DNA sequences of the present invention can be inserted into vectors such as plasmids, cosmids, viral vectors, or yeast artificial chromosomes.
Included in the present invention are DNA sequences that hybridize to SEQ.ll~.NO.:1 under stringent conditions. By way of example, and not limitation, a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA. is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X
SSC, 5X Denhardt's solution, and 100 pg/ml denatured salmon sperm DNA. Filters are hybridized far 12 to 48 hrs at 65°C in prehybridization mixture containing 100 pg/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32P-labeled probe.
Washing of filters is done at 37°C for 1 hr in a solution containing 2X
SSC, 0.1%
For the purposes of this invention;
"Substantially free from other proteins" means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other proteins.
Thus, an HGO1 protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-HGO 1 proteins. Whether a given HGO1 protein preparation is substantially free from other proteins can be determined by such conventional techniques of assessing protein purity as, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting.
"Substantially free from other nucleic acids" means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other nucleic acids. Thus, an HGO1 DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-HGO1 nucleic acids. Whether a given HGO1 DNA preparation is substantially free from other nucleic acids can be determined by such conventional techniques of assessing nucleic acid purity as, e.g., agarose gel electrophoresis combined with appropriate staining methods, e.g., ethidium bromide staining, or by sequencing.
A polypeptide has "substantially the same biological activity" as HGO1 if that polypeptide has a Kd for a ligand that is no more than 5-fold greater than the Kd of HGO 1 for the same ligand.
A "conservative amino acid substitution" refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue.
Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, vaiine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid).
The present invention relates to the identification and cloning ofHG0l, a novel G-protein coupled receptor. The present invention provides DNA
encoding HGO1 that is substantially free from other nucleic acids. The present invention also provides recombinant DNA molecules encoding HGO1.
The present invention provides a DNA molecule substantially free from other nucleic acids having the nucleotide sequence shown in Figure 1 as SEQ.ID.NO.:1. Analysis of SEQ.ID.NO.:1 revealed that it contains an open reading frame at positions 84-1,526. Thus, the present invention also provides a DNA
molecule substantially free from other nucleic acids comprising the nucleotide sequence of positions 84-1,526 of SEQ.117.N0.:1. The present invention also provides recombinant DNA molecules comprising the nucleotide sequence of positions 84-1,526 of SEQ.m.N0.:1.
Sequence analysis of the open reading frame of the HGO1 DNA
revealed that it encodes a protein of 481 amino acids with a predicted signal peptide.
Based on its predicted amino acid sequence, HGO1 most likely represents a novel GPCR. Northern blot analysis showed that HGO1 RNA is highly expressed in the brain and not in the pancreas, kidney, skeletal muscle, liver, lung, placenta, or heart.
Based upon amino acid sequence alignments, HGO1 appears to be related to the endothelin receptors (Figure 4).
The novel DNA sequences of the present invention encoding HGO l, in whole or in part, can be linked with other DNA sequences, i. e., DNA sequences to which HGO1 is not naturally linked, to form "recombinant DNA molecules"
containing HGO1. Such other sequences can include DNA sequences that control transcription or translation such as, e.g., translation initiation sequences, promoters for RNA
polymerase II, transcription or translation termination sequences, enhancer sequences, sequences that control replication in microorganisms, or that confer antibiotic resistance. The novel DNA sequences of the present invention can be inserted into vectors such as plasmids, cosmids, viral vectors, or yeast artificial chromosomes.
Included in the present invention are DNA sequences that hybridize to SEQ.ll~.NO.:1 under stringent conditions. By way of example, and not limitation, a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA. is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X
SSC, 5X Denhardt's solution, and 100 pg/ml denatured salmon sperm DNA. Filters are hybridized far 12 to 48 hrs at 65°C in prehybridization mixture containing 100 pg/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32P-labeled probe.
Washing of filters is done at 37°C for 1 hr in a solution containing 2X
SSC, 0.1%
SDS. This is followed by a wash in O.1X SSC, 0.1% SDS at 50°C for 45 min. before autoradiography.
Other procedures using conditions of high stringency would include either a hybridization step carned out in SXSSC, SX Denhardt's solution, 50%
formamide at 42°C for 12 to 48 hours or a washing step carried out in 0.2X SSPE, 0.2% SDS at 65°C for 30 to 60 minutes.
Reagents mentioned in the foregoing procedures for carrying out high stringency hybridization are well known in the art. Details of the composition of these reagents can be found in, e.g., Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning: A Laborator~r Manual, second edition, Cold Spring Harbor Laboratory Press.
In addition to the foregoing, other conditions of high stringency which may be used are well known in the art.
Another aspect of the present invention includes host cells that have been engineered to contain and/or express DNA sequences encoding HGO1. Such recombinant host cells can be cultured under suitable conditions to produce HGO1.
An expression vector containing DNA encoding HGO 1 can be used for expression of HGO1 in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which are suitable for recombinant expression of HG01 and which are commercially available, include but are not limited to, L cells L-M(TK-) (ATCC
CCL
1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I {ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171 ).
Human embryonic kidney (ILK 293) cells and Chinese hamster ovary (CHO) cells are particularly suitable for expression of the HGO1 protein because these cells express a large number of G-proteins. Thus, it is likely that at least one of these G-proteins will be able to functionally couple the signal generated by interaction of HGO1 and its ligands, thus transmitting this signal to downstream effectors, eventually resulting in a measurable change in some assayable component, e.g., cAMP
level, expression of a reporter gene, hydrolysis of inositol lipids, or intracellular Ca2+ levels.
A variety of mammalian expression vectors can be used to express recombinant HGO1 in mammalian cells. Commercially available mammalian expression vectors which are suitable include, but are not limited to, pMClneo (Stratagene), pSGS (Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), and pSV2-dhfr (ATCC 37146). Following expression in recombinant cells, HGO1 can be purified by conventional techniques to a level that is substantially free from other proteins.
The present invention includes HGO1 protein substantially free from other proteins. The amino acid sequence of the full-length HGO1 protein is shown in Figure 2 as SEQ.1D.N0.:2. Thus, the present invention includes HGO1 protein substantially free from other proteins having the amino acid sequence SEQ.ID.N0.:2.
The present invention also includes HGO1 proteins lacking a signal sequence.
An example of such an HGO1 protein lacking a signal sequence is positions 26-481 of SEQ.ID.N0.:2.
The present invention also includes recombinant DNA encoding an HGO1 protein lacking a signal sequence. Thus, the present invention includes a recombinant DNA molecule encoding an HG01 protein having the amino acid sequence of positions 26-481 of SEQ.ID.N0.:2. The present invention also includes a recombinant DNA molecule comprising the nucleotide sequence of positions 159-1,526 of SEQ.ID.NO.:1.
As with many receptor proteins, it is possible to modify many of the amino acids of HG01, particularly those which are not found in the ligand binding domain, and still retain substantially the same biological activity as the original receptor. Thus this invention includes modified HGO1 polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as HGO1. It is generally accepted that single amino acid substitutions do not usually alter the biological activity of a protein (see, e.g., Molecular Biology of the Gene, Watson et al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science 244:1081-1085). Accordingly, the present invention includes polypeptides where one amino acid substitution has been made in SEQ.117.N0.:2 or in one of the HGO1 polypeptides lacking a signal sequence listed above, wherein the polypeptides still retain substantially the same biological activity as HGO1. The present invention also includes polypeptides where two amino acid substitutions have been made in SEQ.ID.N0.:2 or in one of the HGO1 polypeptides lacking a signal sequence listed above, wherein the polypeptides still retain substantially the same biological activity as HGO1. In particular, the present invention includes embodiments where the above-described substitutions are conservative substitutions. In particular, the present invention includes embodiments where the above-described substitutions do not occur in the ligand-binding domain of HGO 1.
The present invention also includes C-terminal truncated forms of HG01, particularly those which encompass the extracellular portion of the receptor, but lack the intracellular signaling portion of the receptor. Such truncated receptors are useful in various binding assays described herein, for crystallization studies, and for structure-activity-relationship studies.
Romano et al., 1996, J. Biol. Chem. 271:28612-28616 demonstrated that some GPCRs are often found as homodimers formed by an intermolecular disulfide bond. The location of the cysteines responsible for the disulfide bond was found to be in the amino terminal 17kD of the receptors. Accordingly, the present invention includes dimers of HGO 1 proteins.
It has been found that, in some case, membrane spanning regions of receptor proteins can be used to inhibit receptor function (Ng et al., 1996, Biochem.
Biophys. Res. Comm. 227:200-204; Hebert et al., 1996, J. Biol. Chem. 271, 16392; Lofts et al., Oncogene 8:2813-2820). Accordingly, the present invention provides peptides derived from the seven membrane spanning regions of HGO1 and their use to inhibit HGO1 function. Such peptides can include the whole or parts of the receptor membrane spanning domains.
The present invention also includes chimeric HGO1 proteins. Chimeric HGO1 proteins consist of a contiguous polypeptide sequence of HGO 1 fused in frame to a polypeptide sequence of a non-HGO1 protein. For example, the N-terminal domain and seven transmembrane spanning domains of HGO 1 fused at the C-terminus in frame to a G pratein would be a chimeric HGO1 protein.
The present invention also includes HGO1 proteins that are in the form of multimeric structures, e.g., dimers. Such multimers of other G-protein coupled 7_ receptors are known (Hebert et al., 1996, J. Biol. Chem. 271, 16384-16392; Ng et al., 1996, Biochem. Biophys. Res. Comm. 227, 200-204; Romano et al., 1996, J. Biol.
Chem. 271, 28612-28616).
The present invention also includes isolated forms of HG01 proteins.
By "isolated HGO1 protein" is meant HGO1 protein that has been isolated from a natural source. Use of the term "isolated" indicates that HGO1 protein has been removed from its normal cellular environment. Thus, an isolated HGO1 protein may be in a cell-free solution or placed in a different cellular environment from that in which it occurs naturally. The term isolated does not imply that an isolated protein is the only protein present. but instead means that an isolated HGO1 protein is at least 95% free of non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the HGO1 protein. Thus, an HGO 1 protein that is expressed in bacteria or even in eukaryotic cells which do not naturally (i.e., without human intervention) express it through recombinant means is an "isolated HG01 protein."
The specificity of binding of compounds showing affinity for HGO1 is shown by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from these cells. Expression of the cloned receptor and screening for compounds that bind to HGO1 or that inhibit the binding of a known, radiolabeled ligand of HGO 1 to these cells, or membranes prepared from these cells, provides an effective method for the rapid selection of compounds with high affinity for HGO1. Such ligands need not necessarily be radiolabeled but can also be norusotopic compounds that can be used to displace bound radiolabeled compounds or that can be used as activators in functional assays. Compounds identified by the above method are likely to be agonists or antagonists of HGOI and may be peptides, proteins, or non-proteinaceous organic molecules.
Therefore, the present invention includes assays by which HGO1 agonists and antagonists may be identified. Analogous methods for identifying agonists and antagonists of other receptors are well known in the art and can be adapted to identify agonists and antagonists of HGO 1. For example, Cascieri et al., 1992, Molec. Pharmacol. 41:1096-1099 describe a method for identifying substances that inhibit agonist binding to rat neurokinin receptors and thus are potential agonists or antagonists of neurokinin receptors. The method involves transfecting COS
cells with expression vectors containing rat neurokinin receptors, allowing the transfected cells to grow for a time sufficient to allow the neurokinin receptors to be expressed, _g-harvesting the transfected cells and resuspending the cells in assay buffer containing a known radioactively labeled agonist of the neurokinin receptors either in the presence or the absence of the substance, and then measuring the binding of the radioactively labeled known agonist of the neurokinin receptor to the neurokinin receptor.
If the amount of binding of the known agonist is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of the neurokinin receptor.
Accordingly, the present invention includes a method for determining whether a substance is a potential agonist or antagonist of HGO1 that comprises:
(a) transfecting cells with an expression vector encoding HGO1;
(b) allowing the transfected cells to grow for a time sufficient to allow HGO 1 to be expressed;
(c) harvesting the transfected cells and resuspending the cells in the presence of a known labeled agonist of HGO 1 in the presence and in the absence of the 1S substance;
(d) measuring the binding of the labeled agonist to HGO 1; where if the amount of binding of the known agonist is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of HGO1.
In a modification of the above-described method, step (b) is modified in that the cells are stably transfected with the expression vector containing HG01. In another modification of the above-described method, step (c) is modified in that the cells are not harvested and resuspended but rather the radioactively labeled known agonist and the substance are contacted with the cells while the cells are attached to a substratum, e.g., tissue culture plates.
The conditions under which step (c) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions:
e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
In a particular embodiment of the above-described method, the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells. In other embodiments, the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC
CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NH3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I
(ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).
The present invention also includes a method for determining whether a substance is capable of binding to HGO1, i.e., whether the substance is a potential agonist or an antagonist of HG01, where the method comprises:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HGO1 in the cells;
(b) exposing the test cells to the substance;
{c) measuring the amount of binding of the substance to HGO 1;
(d) comparing the amount of binding of the substance to HGO 1 in the test cells with the amount of binding of the substance to control cells that have not been transfected with HGO1;
wherein if the amount of binding of the substance is greater in the test cells as compared to the control cells, the substance is capable of binding to HGO1.
Determining whether the substance is actually an agonist or antagonist can then be accomplished by the use of functional assays such as, e.g., the assay involving the use of promiscuous G-proteins described below.
The conditions under which step (b) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions:
e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
The assays described above can be carried out with cells that have been transiently or stably transfected with HGO1. Transfection is meant to include any method known in the art for introducing HGO1 into the test cells. For example, transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing HGO 1, and electroporation.
Where binding of the substance or agonist to HGO1 is measured, such binding can be measured by employing a labeled substance or agonist. The substance or agonist can be labeled in any convenient manner known to the art, e.g., radioactively, fluorescently, enzymatically.
In particular embodiments of the above-described methods, HG01 has an amino acid sequence selected from the group consisting of SEQ.1D.N0.:2 and positions 26-481 of SEQ.ID.N0.:2;
The above-described methods can be modified in that, rather than exposing the test cells to the substance, membranes can be prepared from the test cells and those membranes can be exposed to the substance. Such a modif cation utilizing membranes rather than cells is well known in the art and is described in, e.g., Hess et al., 1992, Biochem. Biophys. Res. Comm. 184:260-268.
Accordingly, the present invention provides a method for determining whether a substance is capable of binding to HGO1 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HGO 1 in the cells;
(b) preparing membranes containing HGO1 from the test cells and exposing the membranes from the test cells to the substance;
(c) measuring the amount of binding of the substance to the HGO 1 in the membranes from the test cells;
(d) comparing the amount of binding of the substance to HG01 in the membranes from the test cells with the amount of binding of the substance to membranes from control cells that have not been transfected with HGO1;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.N0.:2;
where if the amount of binding of the substance to HGO1 in the membranes from the test cells is greater than the amount of binding of the substance to the membrances from the control cells, then the substance is capable of binding to HGO1.
The present invention provides a method for determining whether a substance is capable of binding to HGO1 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG01 in the cells;
(b) exposing the test cells to a Ggand of HGO1 under conditions such that the ligand binds to the HGO1 in the test cells;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is suspected of being capable of binding to HGOl;
(d) measuring the amount of binding of the ligand to HGO1 in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HGO1 in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HGO1 in the presence of the substance indicates that the substance is capable of binding to HGO1;
where HGO 1 has an amino acid sequence selected from the group consisting of SEQ.ID.N0.:2 and positions 26-481 of SEQ.m.N0.:2.
The present invention provides a method for determining whether a substance is capable of binding to HGO1 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HGOl in the cells;
(b) preparing membranes containing HGO l from the test cells and exposing the membranes to a ligand of HG01 under conditions such that the ligand binds to the HG01 in the membranes;
(c) subsequently or concurrently to step (b), exposing the membranes from the test cells to a substance;
(d) measuring the amount of binding of the ligand to the HGO1 in the membranes in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HGO 1 in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HGO 1 in the membranes in the presence of the substance indicates that the substance is capable of binding to HGO1;
where HGO1 has an amino acid sequence selected from the group consisting of SEQ.m.N0.:2 and positions 26-481 of SEQ.ID.N0.:2.
As a further modification of the above-described methods, RNA
encoding HGO1 can be prepared as, e.g., by in vitro transcription using a plasmid containing HGO 1 under the control of a bacteriophage T7 promoter, and the RNA
can be microinjected into Xenopus oocytes in order to cause the expression of HGO1 in the oocytes. Substances are then tested for binding to the HGO1 expressed in the oocytes.
Alternatively, rather than detecting binding, the effect of the substances on the electrophysiological properties of the oocytes can be determined.
The present invention includes assays by which HGO 1 agonists and antagonists may be identified by their ability to stimulate or antagonize a functional response mediated by HGO1. HGO1 belongs to the class of proteins known as G-protein coupled receptors (GPCRs). GPCRs transmit signals across cell membranes upon the binding of ligand. The ligand-bound GPCR interacts with a heterotrimeric G-protein, causing the Ga subunit of the G-protein to disassociate from the G(3 and Gy subunits. The Ga subunit can then go on to activate a variety of second messenger systems.
Generally, a particular GPCR is only coupled to a particular type of G-protein. Thus, to observe a functional response from the GPCR, it is necessary to ensure that the proper G-protein is present in the system containing the GPCR.
It has been found, however, that there are certain G-proteins that are "promiscuous."
These promiscuous G-proteins will couple to, and thus transduce a functional signal from, virtually any GPCR. See Offermanns & Simon, 1995, J. Biol. Chem. 270:15175, 15180 (Offermanns). Offermanns described a system in which cells are transfected with expression vectors that result in the expression of one of a large number of GPCRs as well as the expression of one of the promiscuous G-proteins GoclS or Gocl6. Upon the addition of an agonist of the GPCR to the transfected cells, the GPCR was activated and was able, via Ga l s or Ga 16, to activate the (3 isoform of phospholipase C, leading to an increase in inositol phosphate levels in the cells.
Therefore, by making use of these promiscuous G-proteins as in Offermanns, it is possible to set up functional assays for HGO1, even in the absence of knowledge of the G-protein with which HGO1 is coupled in vivo. One possibility is to create a fusion or chimeric protein composed of the extracellular and membrane spanning portion of HGO1 fused to a promiscuous G-protein. Such a fusion protein would be expected to transduce a signal following binding of ligand to the portion of the fusion protein. Accordingly, the present invention provides a method of identifying antagonists of HGO 1 comprising:
(a) providing cells that expresses a chimeric HGO1 protein fused at its C-terminus to a promiscuous G-protein;
(b) exposing the cells to an agonist of HGO1;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HGO1;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the presence of the substance as compared to the level of inositol phosphates in the cells in the absence of the substance indicates that the substance is an antagonist of HGO 1.
Another possibility for utilizing promiscuous G-proteins in connection with HGO 1 includes a method of identifying agonists of HGO l comprising:
(a) providing cells that expresses both HGO1 and a promiscuous G-protein;
(b) exposing the cells to a substance that is a suspected agonist of HGO l;
(c) measuring the level of inositol phosphates in the cells;
where an increase in the level of inositol phosphates in the cells as compared to the level of inositol phosphates in the cells in the absence of the suspected agorust indicates that the substance is an agonist of HGOI.
Levels of inositol phosphates can be measured by monitoring calcium mobilization. Intracellular calcium mobilization is typically assayed in whole cells under a microscope using fluorescent dyes or in cell suspensions via luminescence using the aequorin assay.
In a particular embodiment of the above-described method, the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells. In other embodiments, the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC
CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I
(ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).
In a particular embodiment of the above-described method, the cells are transfected with expression vectors that direct the expression of HGO 1 and the promiscuous G protein in the cells.
The conditions under which step (b) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions:
e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about SS°C.
In a particular embodiment of the above-described method, the promiscuous G-protein is selected from the group consisting of Gals or Gocl6.
Expression vectors containing Gals or Gal6 are known in the art. See, e.g., Offermanns; Buhl et al., 1993, FEBS Lett. 323:132-134; Amatruda et ad., 1993, J.
Biol. Chem. 268:10139-10144.
The above-described assay can be easily modified to form a method to 5 identify antagonists of HGO 1. Such a method is also part of the present invention and comprises:
(a) providing cells that expresses both HGO1 and a promiscuous G-protein;
(b) exposing the cells to a substance that is an agonist of HGO1;
10 (c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HGO1;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the presence of the suspected antagonist as compared to the level of inositol phosphates in 15 the cells in the absence of the suspected antagonist indicates that the substance is an antagonist of HGOI .
In a particular embodiment of the above-described method, the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells. In other embodiments, the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC
20 CCL 1.2), 293 (ATCC CRL 1573), Raji {ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 {ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIII/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I
(ATCC CRL 1616), B S-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171 ).
The conditions under which steps (b) and (c) of the method are 25 practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C
to about 55°C.
In a particular embodiment of the above-described method, the cells are 30 transfected with expression vectors that direct the expression of HGO1 and the promiscuous G-protein in the cells.
In a particular embodiment of the above-described method, the promiscuous G-protein is selected from the group consisting of Gals or Gal6.
WO 99/60153 PC'f/US99/10808 In particular embodiments of the above-described methods, HGO 1 has an amino acid sequence selected from the group consisting of SEQ.ID.N0.:2 and positions 26-481 of SEQ.117.N0.:2.
When screening compounds in order to identify potential pharmaceuticals that specifically interact with a target receptor, it is necessary to ensure that the compounds identified are as specific as possible for the target receptor.
To do this, it is necessary to screen the compounds against as wide an array as possible of receptors that are similar to the target receptor. Thus, in order to find compounds that are potential pharmaceuticals that interact with receptor A, it is necessary not only to ensure that the compounds interact with receptor A (the "plus target") and produce the desired pharmacological effect through receptor A, it is also necessary to determine that the compounds do not interact with receptors B, C, D, etc.
(the "minus targets"). In general, as part of a screening program, it is important to have as many minus targets as possible (see Hodgson, 1992, Bio/Technology 10:973-980, at 980). HGO 1 proteins and DNA encoding HG01 proteins have utility in that they can be used as "minus targets" in screens design to identify compounds that specifically interact with other G-protein coupled receptors.
The present invention also includes antibodies to the HGO1 protein.
Such antibodies may be polyclonal antibodies or monoclonal antibodies. The antibodies of the present invention are raised against the entire HGOl protein or against suitable antigenic fragments of the protein that are coupled to suitable carriers, e.g., serum albumin or keyhole limpet hemocyanin, by methods well known in the art.
Methods of identifying suitable antigenic fragments of a protein are known in the art.
See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824-3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications in the Biosciences) 4:181-186.
For the production of polyclonal antibodies, HGO1 protein or an antigenic fragment, coupled to a suitable carrier, is injected on a periodic basis into an appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats, mice. The animals are bled periodically and sera obtained are tested for the presence of antibodies to the injected antigen. The injections can be intramuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant.
For the production of monoclonal antibodies, HGO1 protein or an antigenic fragment, coupled to a suitable carrier, is injected into an appropriate non-human host animal as above for the production of polyclonal antibodies. In the case of monoclonal antibodies, the animal is generally a mouse. The animal's spleen cells are then immortalized, often by fusion with a myeloma cell, as described in Kohler &
Milstein, 1975, Nature 256:495-497. For a fuller description of the production of monoclonal antibodies, see Antibodies: A Laboratory Manual, Harlow & Lane, eds., Cold Spring Harbor Laboratory Press, 1988.
Gene therapy may be used to introduce HGO1 polypeptides into the cells of target organs. Nucleotides encoding HGO 1 polypeptides can be ligated into viral vectors which mediate transfer of the nucleotides by infection of recipient cells.
Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, and polio virus based vectors. Alternatively, nucleotides encoding HGO1 polypeptides can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted transfer using ligand-nucleotide conjugates, lipofection, membrane fusion, or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy. Gene therapy with HGO1 polypeptides will be particularly useful for the treatment of diseases where it is beneficial to elevate HGO1 activity.
The following non-limiting examples are presented to better illustrate the invention.
Cloning and sequencing of HG01 A cDNA fragment encoding full-length HGO 1 can be isolated from a human fetal brain cDNA library by using the polymerise chain reaction (PCR) employing the following primer pair:
HGO1 FL1528R TGGGGAAAGAACAGCACACACC (SEQ. B7. NO.:11) HGO1 FL31F CTGGGCTGGCTGTCTCCTGCTC (SEQ.117. N0.:12) PCR reactions can be carried out with a variety of thermostable enzymes including but not limited to AmpliTaq, AmpliTaq Gold, Vent polymerise.
For AmpliTaq, reactions can be carried out in 10 mM Tris-Cl, pH 8.3, 2.0 mM
MgCl2, 200 pM for each dNTP, 50 mM KCI, 0.2 wM for each primer, 10 ng of DNA
template, 0.05 units/wl of AmpliTaq. The reactions are heated at 95°C
for 3 minutes and then cycled 35 times using the cycling parameters of 95°C, 20 seconds, 62°C, 20 seconds, 72°C, 3 minutes. In addition to these conditions, a variety of suitable PCR
protocols can be found in PCR Primer. A Laborator~Manual, edited by C.W.
Dieffenbach and G.S.Dveksler, 1995, Cold Spring Harbor Laboratory Press.
A suitable cDNA library from which a clone encoding HGO1 can be isolated would be a random primed fetal brain cDNA library consisting of approximately 4.0 million primary clones constructed in the plasmid vector pBluescript (Stratagene, LaJolla, CA). The primary clones of such a library can be subdivided into pools with each pool containing approximately 20,000 clones and each pool can be amplified separately.
By this method, a cDNA fragment encoding an open reading frame of 481 amino acids (SEQ.ID.N0.:2) is obtained. This cDNA fragment can be cloned into a suitable cloning vector or expression vector. For example, the fragment can be cloned into the mammalian expression vector pcDNA3.1 (Invitrogen, San Diego, Ca).
HGO1 protein can then be produced by transferring an expression vector comprising positions 84-1,526 of SEQ.ID.NO.:1 or portions thereof into a suitable host cell and growing the host cell under appropriate conditions. HGO1 protein can then be isolated by methods well known in the art.
As an alternative to the above-described PCR method, a cDNA clone encoding HGO1 can be isolated from a cDNA library using as a probe oligonucleotides specific for HGO1 and methods well known in the art for screening cDNA
libraries with oligonucleotide probes. Such methods are described in, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A
Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I, II. Oligonucleotides that are specific for HGO1 and that can be used to screen cDNA libraries are:
HG01.F223 GCCTGCCCATCCTCTTCACA (SEQ. ID. N0.:3) HGO1.R349 CCACGGTCAGGCCCACCA (SEQ. ID. N0.:4) HGO1.R145 ATACAGGGACTCGGGCAGGC (SEQ. m. N0.:5) HG01.R107 TCCTGGACTGCTGCTGCT (SEQ. B~. N0.:6) HGO1.F715 CGTTGAAGCGGTCAATGC (SEQ. )Z7. N0.:7) HGO1.VW56 0F TGGGGAAAGAACAGCAGACA (SEQ.117. N0.:8) HG01.43R ATGGCTGGATGAGCAGGAGA (SEQ. ID. N0.:9) HG01.85R CCACAGCCAAAATCACAGCA (SEQ. >D. NO.:10) - 1$ -HG01_FL1528R TGGGGAAA.GAACAGCACACACC (SEQ. m. NO.:11) HGO1_FL31F CTGGGCTGGCTGTCTCCTGCTC (SEQ. m. N0.:12) PBS.873F CCCAGGCTTTACACTTTATGCTTCC (SEQ. B7. N0.:13) PBS.543R GGGGATGTGCTGCAAGGCGA (SEQ. DJ. N0.:14) Membrane-spanning proteins, such as GPCRs, when first translated generally possess an approximately 16 to 40 amino acid segment known as a signal sequence. Signal sequences direct the nascent protein to be transported through the endoplasmic reticulum membrane, following which signal sequences are cleaved from the protein. Signal sequences generally contain from 4 to 12 hydrophobic residues but otherwise possess little sequence homology. The Protein Analysis tool of the GCG
program (Genetics Computer Group, Madison, Wisconsin), a computer program capable of identifying likely signal sequences, was used to examine the N
terminus of HGO 1. A likely signal sequence was found at positions 13-25 of (SEQ. m.
N0.:2).
Tissue distribution of HGO1 RNA transcripts An approximately 1.5 kb fragment containing HGO1 was randomly primed with a 32P-dCTP using the Megaprime DNA Labeling System (Amersham) and used to probe a Human MTN Blot (Clontech Cat # 7760-1, Clontech, Palo Alto, CA, USA). The MTN blot was hybridized in Expresshyb (Clontech) containing 2x106 cpm/ml HGO1 probe at 65°C overnight and washed to a final stringency of 0.1 X
SSC/0.5% SDS at 65°C, and then exposed to X-ray film by autoradiongraphy. The results are shown in Figure 3.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.
Other procedures using conditions of high stringency would include either a hybridization step carned out in SXSSC, SX Denhardt's solution, 50%
formamide at 42°C for 12 to 48 hours or a washing step carried out in 0.2X SSPE, 0.2% SDS at 65°C for 30 to 60 minutes.
Reagents mentioned in the foregoing procedures for carrying out high stringency hybridization are well known in the art. Details of the composition of these reagents can be found in, e.g., Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning: A Laborator~r Manual, second edition, Cold Spring Harbor Laboratory Press.
In addition to the foregoing, other conditions of high stringency which may be used are well known in the art.
Another aspect of the present invention includes host cells that have been engineered to contain and/or express DNA sequences encoding HGO1. Such recombinant host cells can be cultured under suitable conditions to produce HGO1.
An expression vector containing DNA encoding HGO 1 can be used for expression of HGO1 in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which are suitable for recombinant expression of HG01 and which are commercially available, include but are not limited to, L cells L-M(TK-) (ATCC
CCL
1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I {ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171 ).
Human embryonic kidney (ILK 293) cells and Chinese hamster ovary (CHO) cells are particularly suitable for expression of the HGO1 protein because these cells express a large number of G-proteins. Thus, it is likely that at least one of these G-proteins will be able to functionally couple the signal generated by interaction of HGO1 and its ligands, thus transmitting this signal to downstream effectors, eventually resulting in a measurable change in some assayable component, e.g., cAMP
level, expression of a reporter gene, hydrolysis of inositol lipids, or intracellular Ca2+ levels.
A variety of mammalian expression vectors can be used to express recombinant HGO1 in mammalian cells. Commercially available mammalian expression vectors which are suitable include, but are not limited to, pMClneo (Stratagene), pSGS (Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC 37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), and pSV2-dhfr (ATCC 37146). Following expression in recombinant cells, HGO1 can be purified by conventional techniques to a level that is substantially free from other proteins.
The present invention includes HGO1 protein substantially free from other proteins. The amino acid sequence of the full-length HGO1 protein is shown in Figure 2 as SEQ.1D.N0.:2. Thus, the present invention includes HGO1 protein substantially free from other proteins having the amino acid sequence SEQ.ID.N0.:2.
The present invention also includes HGO1 proteins lacking a signal sequence.
An example of such an HGO1 protein lacking a signal sequence is positions 26-481 of SEQ.ID.N0.:2.
The present invention also includes recombinant DNA encoding an HGO1 protein lacking a signal sequence. Thus, the present invention includes a recombinant DNA molecule encoding an HG01 protein having the amino acid sequence of positions 26-481 of SEQ.ID.N0.:2. The present invention also includes a recombinant DNA molecule comprising the nucleotide sequence of positions 159-1,526 of SEQ.ID.NO.:1.
As with many receptor proteins, it is possible to modify many of the amino acids of HG01, particularly those which are not found in the ligand binding domain, and still retain substantially the same biological activity as the original receptor. Thus this invention includes modified HGO1 polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as HGO1. It is generally accepted that single amino acid substitutions do not usually alter the biological activity of a protein (see, e.g., Molecular Biology of the Gene, Watson et al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science 244:1081-1085). Accordingly, the present invention includes polypeptides where one amino acid substitution has been made in SEQ.117.N0.:2 or in one of the HGO1 polypeptides lacking a signal sequence listed above, wherein the polypeptides still retain substantially the same biological activity as HGO1. The present invention also includes polypeptides where two amino acid substitutions have been made in SEQ.ID.N0.:2 or in one of the HGO1 polypeptides lacking a signal sequence listed above, wherein the polypeptides still retain substantially the same biological activity as HGO1. In particular, the present invention includes embodiments where the above-described substitutions are conservative substitutions. In particular, the present invention includes embodiments where the above-described substitutions do not occur in the ligand-binding domain of HGO 1.
The present invention also includes C-terminal truncated forms of HG01, particularly those which encompass the extracellular portion of the receptor, but lack the intracellular signaling portion of the receptor. Such truncated receptors are useful in various binding assays described herein, for crystallization studies, and for structure-activity-relationship studies.
Romano et al., 1996, J. Biol. Chem. 271:28612-28616 demonstrated that some GPCRs are often found as homodimers formed by an intermolecular disulfide bond. The location of the cysteines responsible for the disulfide bond was found to be in the amino terminal 17kD of the receptors. Accordingly, the present invention includes dimers of HGO 1 proteins.
It has been found that, in some case, membrane spanning regions of receptor proteins can be used to inhibit receptor function (Ng et al., 1996, Biochem.
Biophys. Res. Comm. 227:200-204; Hebert et al., 1996, J. Biol. Chem. 271, 16392; Lofts et al., Oncogene 8:2813-2820). Accordingly, the present invention provides peptides derived from the seven membrane spanning regions of HGO1 and their use to inhibit HGO1 function. Such peptides can include the whole or parts of the receptor membrane spanning domains.
The present invention also includes chimeric HGO1 proteins. Chimeric HGO1 proteins consist of a contiguous polypeptide sequence of HGO 1 fused in frame to a polypeptide sequence of a non-HGO1 protein. For example, the N-terminal domain and seven transmembrane spanning domains of HGO 1 fused at the C-terminus in frame to a G pratein would be a chimeric HGO1 protein.
The present invention also includes HGO1 proteins that are in the form of multimeric structures, e.g., dimers. Such multimers of other G-protein coupled 7_ receptors are known (Hebert et al., 1996, J. Biol. Chem. 271, 16384-16392; Ng et al., 1996, Biochem. Biophys. Res. Comm. 227, 200-204; Romano et al., 1996, J. Biol.
Chem. 271, 28612-28616).
The present invention also includes isolated forms of HG01 proteins.
By "isolated HGO1 protein" is meant HGO1 protein that has been isolated from a natural source. Use of the term "isolated" indicates that HGO1 protein has been removed from its normal cellular environment. Thus, an isolated HGO1 protein may be in a cell-free solution or placed in a different cellular environment from that in which it occurs naturally. The term isolated does not imply that an isolated protein is the only protein present. but instead means that an isolated HGO1 protein is at least 95% free of non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the HGO1 protein. Thus, an HGO 1 protein that is expressed in bacteria or even in eukaryotic cells which do not naturally (i.e., without human intervention) express it through recombinant means is an "isolated HG01 protein."
The specificity of binding of compounds showing affinity for HGO1 is shown by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from these cells. Expression of the cloned receptor and screening for compounds that bind to HGO1 or that inhibit the binding of a known, radiolabeled ligand of HGO 1 to these cells, or membranes prepared from these cells, provides an effective method for the rapid selection of compounds with high affinity for HGO1. Such ligands need not necessarily be radiolabeled but can also be norusotopic compounds that can be used to displace bound radiolabeled compounds or that can be used as activators in functional assays. Compounds identified by the above method are likely to be agonists or antagonists of HGOI and may be peptides, proteins, or non-proteinaceous organic molecules.
Therefore, the present invention includes assays by which HGO1 agonists and antagonists may be identified. Analogous methods for identifying agonists and antagonists of other receptors are well known in the art and can be adapted to identify agonists and antagonists of HGO 1. For example, Cascieri et al., 1992, Molec. Pharmacol. 41:1096-1099 describe a method for identifying substances that inhibit agonist binding to rat neurokinin receptors and thus are potential agonists or antagonists of neurokinin receptors. The method involves transfecting COS
cells with expression vectors containing rat neurokinin receptors, allowing the transfected cells to grow for a time sufficient to allow the neurokinin receptors to be expressed, _g-harvesting the transfected cells and resuspending the cells in assay buffer containing a known radioactively labeled agonist of the neurokinin receptors either in the presence or the absence of the substance, and then measuring the binding of the radioactively labeled known agonist of the neurokinin receptor to the neurokinin receptor.
If the amount of binding of the known agonist is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of the neurokinin receptor.
Accordingly, the present invention includes a method for determining whether a substance is a potential agonist or antagonist of HGO1 that comprises:
(a) transfecting cells with an expression vector encoding HGO1;
(b) allowing the transfected cells to grow for a time sufficient to allow HGO 1 to be expressed;
(c) harvesting the transfected cells and resuspending the cells in the presence of a known labeled agonist of HGO 1 in the presence and in the absence of the 1S substance;
(d) measuring the binding of the labeled agonist to HGO 1; where if the amount of binding of the known agonist is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of HGO1.
In a modification of the above-described method, step (b) is modified in that the cells are stably transfected with the expression vector containing HG01. In another modification of the above-described method, step (c) is modified in that the cells are not harvested and resuspended but rather the radioactively labeled known agonist and the substance are contacted with the cells while the cells are attached to a substratum, e.g., tissue culture plates.
The conditions under which step (c) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions:
e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
In a particular embodiment of the above-described method, the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells. In other embodiments, the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC
CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NH3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I
(ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).
The present invention also includes a method for determining whether a substance is capable of binding to HGO1, i.e., whether the substance is a potential agonist or an antagonist of HG01, where the method comprises:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HGO1 in the cells;
(b) exposing the test cells to the substance;
{c) measuring the amount of binding of the substance to HGO 1;
(d) comparing the amount of binding of the substance to HGO 1 in the test cells with the amount of binding of the substance to control cells that have not been transfected with HGO1;
wherein if the amount of binding of the substance is greater in the test cells as compared to the control cells, the substance is capable of binding to HGO1.
Determining whether the substance is actually an agonist or antagonist can then be accomplished by the use of functional assays such as, e.g., the assay involving the use of promiscuous G-proteins described below.
The conditions under which step (b) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions:
e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
The assays described above can be carried out with cells that have been transiently or stably transfected with HGO1. Transfection is meant to include any method known in the art for introducing HGO1 into the test cells. For example, transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing HGO 1, and electroporation.
Where binding of the substance or agonist to HGO1 is measured, such binding can be measured by employing a labeled substance or agonist. The substance or agonist can be labeled in any convenient manner known to the art, e.g., radioactively, fluorescently, enzymatically.
In particular embodiments of the above-described methods, HG01 has an amino acid sequence selected from the group consisting of SEQ.1D.N0.:2 and positions 26-481 of SEQ.ID.N0.:2;
The above-described methods can be modified in that, rather than exposing the test cells to the substance, membranes can be prepared from the test cells and those membranes can be exposed to the substance. Such a modif cation utilizing membranes rather than cells is well known in the art and is described in, e.g., Hess et al., 1992, Biochem. Biophys. Res. Comm. 184:260-268.
Accordingly, the present invention provides a method for determining whether a substance is capable of binding to HGO1 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HGO 1 in the cells;
(b) preparing membranes containing HGO1 from the test cells and exposing the membranes from the test cells to the substance;
(c) measuring the amount of binding of the substance to the HGO 1 in the membranes from the test cells;
(d) comparing the amount of binding of the substance to HG01 in the membranes from the test cells with the amount of binding of the substance to membranes from control cells that have not been transfected with HGO1;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.N0.:2;
where if the amount of binding of the substance to HGO1 in the membranes from the test cells is greater than the amount of binding of the substance to the membrances from the control cells, then the substance is capable of binding to HGO1.
The present invention provides a method for determining whether a substance is capable of binding to HGO1 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG01 in the cells;
(b) exposing the test cells to a Ggand of HGO1 under conditions such that the ligand binds to the HGO1 in the test cells;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is suspected of being capable of binding to HGOl;
(d) measuring the amount of binding of the ligand to HGO1 in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HGO1 in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HGO1 in the presence of the substance indicates that the substance is capable of binding to HGO1;
where HGO 1 has an amino acid sequence selected from the group consisting of SEQ.ID.N0.:2 and positions 26-481 of SEQ.m.N0.:2.
The present invention provides a method for determining whether a substance is capable of binding to HGO1 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HGOl in the cells;
(b) preparing membranes containing HGO l from the test cells and exposing the membranes to a ligand of HG01 under conditions such that the ligand binds to the HG01 in the membranes;
(c) subsequently or concurrently to step (b), exposing the membranes from the test cells to a substance;
(d) measuring the amount of binding of the ligand to the HGO1 in the membranes in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HGO 1 in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HGO 1 in the membranes in the presence of the substance indicates that the substance is capable of binding to HGO1;
where HGO1 has an amino acid sequence selected from the group consisting of SEQ.m.N0.:2 and positions 26-481 of SEQ.ID.N0.:2.
As a further modification of the above-described methods, RNA
encoding HGO1 can be prepared as, e.g., by in vitro transcription using a plasmid containing HGO 1 under the control of a bacteriophage T7 promoter, and the RNA
can be microinjected into Xenopus oocytes in order to cause the expression of HGO1 in the oocytes. Substances are then tested for binding to the HGO1 expressed in the oocytes.
Alternatively, rather than detecting binding, the effect of the substances on the electrophysiological properties of the oocytes can be determined.
The present invention includes assays by which HGO 1 agonists and antagonists may be identified by their ability to stimulate or antagonize a functional response mediated by HGO1. HGO1 belongs to the class of proteins known as G-protein coupled receptors (GPCRs). GPCRs transmit signals across cell membranes upon the binding of ligand. The ligand-bound GPCR interacts with a heterotrimeric G-protein, causing the Ga subunit of the G-protein to disassociate from the G(3 and Gy subunits. The Ga subunit can then go on to activate a variety of second messenger systems.
Generally, a particular GPCR is only coupled to a particular type of G-protein. Thus, to observe a functional response from the GPCR, it is necessary to ensure that the proper G-protein is present in the system containing the GPCR.
It has been found, however, that there are certain G-proteins that are "promiscuous."
These promiscuous G-proteins will couple to, and thus transduce a functional signal from, virtually any GPCR. See Offermanns & Simon, 1995, J. Biol. Chem. 270:15175, 15180 (Offermanns). Offermanns described a system in which cells are transfected with expression vectors that result in the expression of one of a large number of GPCRs as well as the expression of one of the promiscuous G-proteins GoclS or Gocl6. Upon the addition of an agonist of the GPCR to the transfected cells, the GPCR was activated and was able, via Ga l s or Ga 16, to activate the (3 isoform of phospholipase C, leading to an increase in inositol phosphate levels in the cells.
Therefore, by making use of these promiscuous G-proteins as in Offermanns, it is possible to set up functional assays for HGO1, even in the absence of knowledge of the G-protein with which HGO1 is coupled in vivo. One possibility is to create a fusion or chimeric protein composed of the extracellular and membrane spanning portion of HGO1 fused to a promiscuous G-protein. Such a fusion protein would be expected to transduce a signal following binding of ligand to the portion of the fusion protein. Accordingly, the present invention provides a method of identifying antagonists of HGO 1 comprising:
(a) providing cells that expresses a chimeric HGO1 protein fused at its C-terminus to a promiscuous G-protein;
(b) exposing the cells to an agonist of HGO1;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HGO1;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the presence of the substance as compared to the level of inositol phosphates in the cells in the absence of the substance indicates that the substance is an antagonist of HGO 1.
Another possibility for utilizing promiscuous G-proteins in connection with HGO 1 includes a method of identifying agonists of HGO l comprising:
(a) providing cells that expresses both HGO1 and a promiscuous G-protein;
(b) exposing the cells to a substance that is a suspected agonist of HGO l;
(c) measuring the level of inositol phosphates in the cells;
where an increase in the level of inositol phosphates in the cells as compared to the level of inositol phosphates in the cells in the absence of the suspected agorust indicates that the substance is an agonist of HGOI.
Levels of inositol phosphates can be measured by monitoring calcium mobilization. Intracellular calcium mobilization is typically assayed in whole cells under a microscope using fluorescent dyes or in cell suspensions via luminescence using the aequorin assay.
In a particular embodiment of the above-described method, the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells. In other embodiments, the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC
CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I
(ATCC CRL 1616), BS-C-1 (ATCC CCL 26) or MRC-5 (ATCC CCL 171).
In a particular embodiment of the above-described method, the cells are transfected with expression vectors that direct the expression of HGO 1 and the promiscuous G protein in the cells.
The conditions under which step (b) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions:
e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about SS°C.
In a particular embodiment of the above-described method, the promiscuous G-protein is selected from the group consisting of Gals or Gocl6.
Expression vectors containing Gals or Gal6 are known in the art. See, e.g., Offermanns; Buhl et al., 1993, FEBS Lett. 323:132-134; Amatruda et ad., 1993, J.
Biol. Chem. 268:10139-10144.
The above-described assay can be easily modified to form a method to 5 identify antagonists of HGO 1. Such a method is also part of the present invention and comprises:
(a) providing cells that expresses both HGO1 and a promiscuous G-protein;
(b) exposing the cells to a substance that is an agonist of HGO1;
10 (c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HGO1;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the presence of the suspected antagonist as compared to the level of inositol phosphates in 15 the cells in the absence of the suspected antagonist indicates that the substance is an antagonist of HGOI .
In a particular embodiment of the above-described method, the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells. In other embodiments, the cells are L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC
20 CCL 1.2), 293 (ATCC CRL 1573), Raji {ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 {ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIII/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I
(ATCC CRL 1616), B S-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171 ).
The conditions under which steps (b) and (c) of the method are 25 practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C
to about 55°C.
In a particular embodiment of the above-described method, the cells are 30 transfected with expression vectors that direct the expression of HGO1 and the promiscuous G-protein in the cells.
In a particular embodiment of the above-described method, the promiscuous G-protein is selected from the group consisting of Gals or Gal6.
WO 99/60153 PC'f/US99/10808 In particular embodiments of the above-described methods, HGO 1 has an amino acid sequence selected from the group consisting of SEQ.ID.N0.:2 and positions 26-481 of SEQ.117.N0.:2.
When screening compounds in order to identify potential pharmaceuticals that specifically interact with a target receptor, it is necessary to ensure that the compounds identified are as specific as possible for the target receptor.
To do this, it is necessary to screen the compounds against as wide an array as possible of receptors that are similar to the target receptor. Thus, in order to find compounds that are potential pharmaceuticals that interact with receptor A, it is necessary not only to ensure that the compounds interact with receptor A (the "plus target") and produce the desired pharmacological effect through receptor A, it is also necessary to determine that the compounds do not interact with receptors B, C, D, etc.
(the "minus targets"). In general, as part of a screening program, it is important to have as many minus targets as possible (see Hodgson, 1992, Bio/Technology 10:973-980, at 980). HGO 1 proteins and DNA encoding HG01 proteins have utility in that they can be used as "minus targets" in screens design to identify compounds that specifically interact with other G-protein coupled receptors.
The present invention also includes antibodies to the HGO1 protein.
Such antibodies may be polyclonal antibodies or monoclonal antibodies. The antibodies of the present invention are raised against the entire HGOl protein or against suitable antigenic fragments of the protein that are coupled to suitable carriers, e.g., serum albumin or keyhole limpet hemocyanin, by methods well known in the art.
Methods of identifying suitable antigenic fragments of a protein are known in the art.
See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824-3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications in the Biosciences) 4:181-186.
For the production of polyclonal antibodies, HGO1 protein or an antigenic fragment, coupled to a suitable carrier, is injected on a periodic basis into an appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats, mice. The animals are bled periodically and sera obtained are tested for the presence of antibodies to the injected antigen. The injections can be intramuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant.
For the production of monoclonal antibodies, HGO1 protein or an antigenic fragment, coupled to a suitable carrier, is injected into an appropriate non-human host animal as above for the production of polyclonal antibodies. In the case of monoclonal antibodies, the animal is generally a mouse. The animal's spleen cells are then immortalized, often by fusion with a myeloma cell, as described in Kohler &
Milstein, 1975, Nature 256:495-497. For a fuller description of the production of monoclonal antibodies, see Antibodies: A Laboratory Manual, Harlow & Lane, eds., Cold Spring Harbor Laboratory Press, 1988.
Gene therapy may be used to introduce HGO1 polypeptides into the cells of target organs. Nucleotides encoding HGO 1 polypeptides can be ligated into viral vectors which mediate transfer of the nucleotides by infection of recipient cells.
Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, and polio virus based vectors. Alternatively, nucleotides encoding HGO1 polypeptides can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted transfer using ligand-nucleotide conjugates, lipofection, membrane fusion, or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy. Gene therapy with HGO1 polypeptides will be particularly useful for the treatment of diseases where it is beneficial to elevate HGO1 activity.
The following non-limiting examples are presented to better illustrate the invention.
Cloning and sequencing of HG01 A cDNA fragment encoding full-length HGO 1 can be isolated from a human fetal brain cDNA library by using the polymerise chain reaction (PCR) employing the following primer pair:
HGO1 FL1528R TGGGGAAAGAACAGCACACACC (SEQ. B7. NO.:11) HGO1 FL31F CTGGGCTGGCTGTCTCCTGCTC (SEQ.117. N0.:12) PCR reactions can be carried out with a variety of thermostable enzymes including but not limited to AmpliTaq, AmpliTaq Gold, Vent polymerise.
For AmpliTaq, reactions can be carried out in 10 mM Tris-Cl, pH 8.3, 2.0 mM
MgCl2, 200 pM for each dNTP, 50 mM KCI, 0.2 wM for each primer, 10 ng of DNA
template, 0.05 units/wl of AmpliTaq. The reactions are heated at 95°C
for 3 minutes and then cycled 35 times using the cycling parameters of 95°C, 20 seconds, 62°C, 20 seconds, 72°C, 3 minutes. In addition to these conditions, a variety of suitable PCR
protocols can be found in PCR Primer. A Laborator~Manual, edited by C.W.
Dieffenbach and G.S.Dveksler, 1995, Cold Spring Harbor Laboratory Press.
A suitable cDNA library from which a clone encoding HGO1 can be isolated would be a random primed fetal brain cDNA library consisting of approximately 4.0 million primary clones constructed in the plasmid vector pBluescript (Stratagene, LaJolla, CA). The primary clones of such a library can be subdivided into pools with each pool containing approximately 20,000 clones and each pool can be amplified separately.
By this method, a cDNA fragment encoding an open reading frame of 481 amino acids (SEQ.ID.N0.:2) is obtained. This cDNA fragment can be cloned into a suitable cloning vector or expression vector. For example, the fragment can be cloned into the mammalian expression vector pcDNA3.1 (Invitrogen, San Diego, Ca).
HGO1 protein can then be produced by transferring an expression vector comprising positions 84-1,526 of SEQ.ID.NO.:1 or portions thereof into a suitable host cell and growing the host cell under appropriate conditions. HGO1 protein can then be isolated by methods well known in the art.
As an alternative to the above-described PCR method, a cDNA clone encoding HGO1 can be isolated from a cDNA library using as a probe oligonucleotides specific for HGO1 and methods well known in the art for screening cDNA
libraries with oligonucleotide probes. Such methods are described in, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A
Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I, II. Oligonucleotides that are specific for HGO1 and that can be used to screen cDNA libraries are:
HG01.F223 GCCTGCCCATCCTCTTCACA (SEQ. ID. N0.:3) HGO1.R349 CCACGGTCAGGCCCACCA (SEQ. ID. N0.:4) HGO1.R145 ATACAGGGACTCGGGCAGGC (SEQ. m. N0.:5) HG01.R107 TCCTGGACTGCTGCTGCT (SEQ. B~. N0.:6) HGO1.F715 CGTTGAAGCGGTCAATGC (SEQ. )Z7. N0.:7) HGO1.VW56 0F TGGGGAAAGAACAGCAGACA (SEQ.117. N0.:8) HG01.43R ATGGCTGGATGAGCAGGAGA (SEQ. ID. N0.:9) HG01.85R CCACAGCCAAAATCACAGCA (SEQ. >D. NO.:10) - 1$ -HG01_FL1528R TGGGGAAA.GAACAGCACACACC (SEQ. m. NO.:11) HGO1_FL31F CTGGGCTGGCTGTCTCCTGCTC (SEQ. m. N0.:12) PBS.873F CCCAGGCTTTACACTTTATGCTTCC (SEQ. B7. N0.:13) PBS.543R GGGGATGTGCTGCAAGGCGA (SEQ. DJ. N0.:14) Membrane-spanning proteins, such as GPCRs, when first translated generally possess an approximately 16 to 40 amino acid segment known as a signal sequence. Signal sequences direct the nascent protein to be transported through the endoplasmic reticulum membrane, following which signal sequences are cleaved from the protein. Signal sequences generally contain from 4 to 12 hydrophobic residues but otherwise possess little sequence homology. The Protein Analysis tool of the GCG
program (Genetics Computer Group, Madison, Wisconsin), a computer program capable of identifying likely signal sequences, was used to examine the N
terminus of HGO 1. A likely signal sequence was found at positions 13-25 of (SEQ. m.
N0.:2).
Tissue distribution of HGO1 RNA transcripts An approximately 1.5 kb fragment containing HGO1 was randomly primed with a 32P-dCTP using the Megaprime DNA Labeling System (Amersham) and used to probe a Human MTN Blot (Clontech Cat # 7760-1, Clontech, Palo Alto, CA, USA). The MTN blot was hybridized in Expresshyb (Clontech) containing 2x106 cpm/ml HGO1 probe at 65°C overnight and washed to a final stringency of 0.1 X
SSC/0.5% SDS at 65°C, and then exposed to X-ray film by autoradiongraphy. The results are shown in Figure 3.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.
Claims (21)
1. A recombinant DNA molecule encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ.ID.N0.:2 and positions 26-481 of SEQ.ID.NO.:2.
2. A recombinant DNA molecule comprising a nucleotide sequence selected from the group consisting of SEQ.ID.NO.:1 and positions 159-1,526 of SEQ.ID.NO.:1.
3. A DNA molecule that hybridizes under stringent conditions to DNA having the nucleotide sequence SEQ.ID.NO.:1.
4. An expression vector comprising the DNA of claim 1.
5. A recombinant host cell comprising the DNA of claim 1.
6. An HG01 protein, substantially free from other proteins, having an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
7. The HG01 protein of claim 6 containing a single amino acid substitution.
8. The HG01 protein of claim 7 where the substitution is a conservative substitution.
9. The HG01 protein of claim 6 containing two amino acid substitutions.
10. A method for determining whether a substance is capable of binding to HG01 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG01 in the cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of binding of the substance to HG01;
(d) comparing the amount of binding of the substance to HG01 in the test cells with the amount of binding of the substance to control cells that have not been transfected with HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2;
where if the amount of binding of the substance to HG01 in the test cells is greater than the amount of binding of the substance to the control cells, then the substance is capable of binding to HG01.
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG01 in the cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of binding of the substance to HG01;
(d) comparing the amount of binding of the substance to HG01 in the test cells with the amount of binding of the substance to control cells that have not been transfected with HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2;
where if the amount of binding of the substance to HG01 in the test cells is greater than the amount of binding of the substance to the control cells, then the substance is capable of binding to HG01.
11. A method for determining whether a substance is capable of binding to HG01 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG01 in the cells;
(b) preparing membranes containing HG01 from the test cells and exposing the membranes from the test cells to the substance;
(c) measuring the amount of binding of the substance to the HG01 in the membranes from the test cells;
(d) comparing the amount of binding of the substance to HG01 in the membranes from the test cells with the amount of binding of the substance to membranes from control cells that have not been transfected with HG01;
where HG01 has an amino acid sequence selected from the goup consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2;
where if the amount of binding of the substance to HG01 in the membranes from the test cells is greater than the amount of binding of the substance to the membrances from the control cells, then the substance is capable of binding to HG01.
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG01 in the cells;
(b) preparing membranes containing HG01 from the test cells and exposing the membranes from the test cells to the substance;
(c) measuring the amount of binding of the substance to the HG01 in the membranes from the test cells;
(d) comparing the amount of binding of the substance to HG01 in the membranes from the test cells with the amount of binding of the substance to membranes from control cells that have not been transfected with HG01;
where HG01 has an amino acid sequence selected from the goup consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2;
where if the amount of binding of the substance to HG01 in the membranes from the test cells is greater than the amount of binding of the substance to the membrances from the control cells, then the substance is capable of binding to HG01.
12. A method for determining whether a substance is capable of binding to HG01 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG01 in the cells;
(b) exposing the test cells to a ligand of HG01 under conditions such that the ligand binds to the HG01 in the test cells;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is suspected of being capable of binding to HG01;
(d) measuring the amount of binding of the ligand to HG01 in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HG01 in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HG01 in the presence of the substance indicates that the substance is capable of binding to HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG01 in the cells;
(b) exposing the test cells to a ligand of HG01 under conditions such that the ligand binds to the HG01 in the test cells;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is suspected of being capable of binding to HG01;
(d) measuring the amount of binding of the ligand to HG01 in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HG01 in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HG01 in the presence of the substance indicates that the substance is capable of binding to HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
13. A method for determining whether a substance is capable of binding to HG01 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG01 in the cells;
(b) preparing membranes containing HG01 from the test cells and exposing the membranes to a ligand of HG01 under conditions such that the ligand binds to the HG01 in the membranes;
(c) subsequently or concurrently to step (b), exposing the membranes from the test cells to a substance;
(d) measuring the amount of binding of the ligand to the HG01 in the membranes in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HG01 in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HG01 in the membranes in the presence of the substance indicates that the substance is capable of binding to HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG01 in the cells;
(b) preparing membranes containing HG01 from the test cells and exposing the membranes to a ligand of HG01 under conditions such that the ligand binds to the HG01 in the membranes;
(c) subsequently or concurrently to step (b), exposing the membranes from the test cells to a substance;
(d) measuring the amount of binding of the ligand to the HG01 in the membranes in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HG01 in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HG01 in the membranes in the presence of the substance indicates that the substance is capable of binding to HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
14. A method for determining whether a substance is a potential agonist or antagonist of HG01 that comprises:
(a) transfecting cells with an expression vector containing HG01;
(b) allowing the transfected cells to grow for a time sufficient to allow HG01 to be expressed;
(c) harvesting the transfected cells and resuspending the cells in assay buffer containing a known labeled agonist of HG01 in the presence and in the absence of the substance;
(d) measuring the binding of the known labeled agonist to HG01;
where if the amount of binding of the known labeled agonist is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
(a) transfecting cells with an expression vector containing HG01;
(b) allowing the transfected cells to grow for a time sufficient to allow HG01 to be expressed;
(c) harvesting the transfected cells and resuspending the cells in assay buffer containing a known labeled agonist of HG01 in the presence and in the absence of the substance;
(d) measuring the binding of the known labeled agonist to HG01;
where if the amount of binding of the known labeled agonist is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
15. A method of identifying agonists of HG01 comprising:
(a) providing cells that expresses both HG01 and a promiscuous G-protein;
(b) exposing the cells to a substance that is a suspected agonist of HG01;
(c) measuring the level of inositol phosphates in the cells;
where an increase in the level of inositol phosphates in the cells in the presence of the substance as compared to the level of inositol phosphates in the cells in the absence of the substance indicates that the substance is an agonist of HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
(a) providing cells that expresses both HG01 and a promiscuous G-protein;
(b) exposing the cells to a substance that is a suspected agonist of HG01;
(c) measuring the level of inositol phosphates in the cells;
where an increase in the level of inositol phosphates in the cells in the presence of the substance as compared to the level of inositol phosphates in the cells in the absence of the substance indicates that the substance is an agonist of HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
16. A method identifying antagonists of HG01 comprising:
(a) providing cells that expresses both HG01 and a promiscuous G-protein;
(b) exposing the cells to an agonist of HG01;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG01;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the presence of the substance as compared to the level of inositol phosphates in the cells in the absence of the substance indicates that the substance is an antagonist of HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
(a) providing cells that expresses both HG01 and a promiscuous G-protein;
(b) exposing the cells to an agonist of HG01;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG01;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the presence of the substance as compared to the level of inositol phosphates in the cells in the absence of the substance indicates that the substance is an antagonist of HG01;
where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
17. A method of identifying antagonists of HG01 comprising:
(a) providing cells that expresses a chimeric HG01 protein fused at its C-terminus to a promiscuous G-protein;
(b) exposing the cells to an agonist of HG01;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG01;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the presence of the substance as compared to the level of inositol phosphates in the cells in the absence of the substance indicates that the substance is an antagonist of HGO1;
where the HG01 protein has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
(a) providing cells that expresses a chimeric HG01 protein fused at its C-terminus to a promiscuous G-protein;
(b) exposing the cells to an agonist of HG01;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG01;
(d) measuring the level of inositol phosphates in the cells;
where a decrease in the level of inositol phosphates in the cells in the presence of the substance as compared to the level of inositol phosphates in the cells in the absence of the substance indicates that the substance is an antagonist of HGO1;
where the HG01 protein has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
18. An antibody that binds specifically to HG01 where HG01 has an amino acid sequence selected from the group consisting of SEQ.ID.NO.:2 and positions 26-481 of SEQ.ID.NO.:2.
19. A method of expressing a truncated version of HG01 protein comprising:
(a) transfecting a host cell with a expression vector that encodes an HG01 protein that has been truncated at the amino terminus;
(b) culturing the transfected cells of step (a) under conditions such that the truncated HG01 protein is expressed.
(a) transfecting a host cell with a expression vector that encodes an HG01 protein that has been truncated at the amino terminus;
(b) culturing the transfected cells of step (a) under conditions such that the truncated HG01 protein is expressed.
20. The method of claim 18 where the truncated HG01 protein is amino acids 26-481.
21. The method of claim 19 where the truncated HG01 protein is a chimeric HG01 protein.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US8629498P | 1998-05-21 | 1998-05-21 | |
| US60/086,294 | 1998-05-21 | ||
| PCT/US1999/010808 WO1999060153A2 (en) | 1998-05-21 | 1999-05-17 | Novel g-protein coupled receptor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2329153A1 true CA2329153A1 (en) | 1999-11-25 |
Family
ID=22197612
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002329153A Abandoned CA2329153A1 (en) | 1998-05-21 | 1999-05-17 | Novel g-protein coupled receptor |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP1082450A4 (en) |
| JP (1) | JP2002515259A (en) |
| CA (1) | CA2329153A1 (en) |
| WO (1) | WO1999060153A2 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10127289A (en) * | 1996-10-29 | 1998-05-19 | Takeda Chem Ind Ltd | New g protein conjugate type receptor protein and its dna |
| DE19805351A1 (en) * | 1998-02-11 | 1999-08-12 | Basf Ag | New human G-protein coupled receptor from brain tissue, used to treat nervous system disorders, e.g. Alzheimer's disease, eating disorders and as cerebral protectant |
-
1999
- 1999-05-17 CA CA002329153A patent/CA2329153A1/en not_active Abandoned
- 1999-05-17 EP EP99924275A patent/EP1082450A4/en not_active Withdrawn
- 1999-05-17 JP JP2000549759A patent/JP2002515259A/en active Pending
- 1999-05-17 WO PCT/US1999/010808 patent/WO1999060153A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP1082450A4 (en) | 2003-01-29 |
| JP2002515259A (en) | 2002-05-28 |
| EP1082450A1 (en) | 2001-03-14 |
| WO1999060153A2 (en) | 1999-11-25 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDC | Discontinued application reinstated | ||
| FZDE | Discontinued |