CA2339347A1 - Novel g protein-coupled receptor cdna sequence - Google Patents
Novel g protein-coupled receptor cdna sequence Download PDFInfo
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Abstract
cDNA encoding a novel human G-protein coupled receptor, HG03, as well as the protein encoded by the cDNA, is provided. Methods of identifying agonists an d antagonists of HG03 are also provided.
Description
w0 00/08133 PCT/US99/17388 TITLE OF THE INVEN7.'ION
NOVEL G PROTEIN-CC)UPLED RECEPTOR cDNA SEQUENCE
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT RBGARD~ING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVEN'JCION
This invention relates to a novel human cDNA encoding a G protein-coupled receptor (GPCR) having homology to members of a family ofreceptors for nucleotides and platelet-activating factor, the protein encoded by the cDNA, and methods of identifying selective agonists and antagonists.
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 ofheterotrimeric 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 ø
and y subunits. The freed a subunit then interacts with other cellular components, and in the process passes on the extracellular signal represented by the presence of the agonist. Occasionally, it is the freed ~i and y 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 extracel.lular amino terminus, and an intracellular - WO 00/0$133 PCT/U899/17388 carboxy terminus (StradE;r et al., 1994, Ann. Rev. Biochem. 63:101-132;
Schertler 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.~~., 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 Stad.el 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 IhIVENTION
The present invention is directed to a novel human cDNA that encodes a G-protein coupled receptor, HG03. The DNA encoding HG03 is substantially free from other nucleic acids and has the nucleotide sequence shown in SEQ.ID.NO.:1.
Also provided is an HG03 protein encoded by the novel cDNA sequence. The HG03 protein is substantially free from other proteins and has the amino acid sequence shown in SEQ.1D.N0.:2. Methods of expressing HG03 in recombinant systems and of identifying agoni.sts and antagonists of HG03 are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the complete cDNA sequence of HG03 (SEQ.ID.NO.: I }.
Figure 2 show the complete amino acid sequence of HG03 (SEQ.ID.:2).
Figure 3A-C shows the translation of HG03 open reading frame. The nucleotide sequence shown is (SEQ.ID.:1). The amino acid sequence shown is (SEQ.ID.:2).
Figure 4 shows the results of a Northern blot of the expression of HG03 mRNA in various human tissues.
Figure 5 shows the alignment of the amino acid sequence of HG03 with the amino acid sequence of the human platelet activating factor receptor (SEQ.ID.:3).
NOVEL G PROTEIN-CC)UPLED RECEPTOR cDNA SEQUENCE
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT RBGARD~ING FEDERALLY-SPONSORED R&D
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVEN'JCION
This invention relates to a novel human cDNA encoding a G protein-coupled receptor (GPCR) having homology to members of a family ofreceptors for nucleotides and platelet-activating factor, the protein encoded by the cDNA, and methods of identifying selective agonists and antagonists.
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 ofheterotrimeric 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 ø
and y subunits. The freed a subunit then interacts with other cellular components, and in the process passes on the extracellular signal represented by the presence of the agonist. Occasionally, it is the freed ~i and y 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 extracel.lular amino terminus, and an intracellular - WO 00/0$133 PCT/U899/17388 carboxy terminus (StradE;r et al., 1994, Ann. Rev. Biochem. 63:101-132;
Schertler 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.~~., 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 Stad.el 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 IhIVENTION
The present invention is directed to a novel human cDNA that encodes a G-protein coupled receptor, HG03. The DNA encoding HG03 is substantially free from other nucleic acids and has the nucleotide sequence shown in SEQ.ID.NO.:1.
Also provided is an HG03 protein encoded by the novel cDNA sequence. The HG03 protein is substantially free from other proteins and has the amino acid sequence shown in SEQ.1D.N0.:2. Methods of expressing HG03 in recombinant systems and of identifying agoni.sts and antagonists of HG03 are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the complete cDNA sequence of HG03 (SEQ.ID.NO.: I }.
Figure 2 show the complete amino acid sequence of HG03 (SEQ.ID.:2).
Figure 3A-C shows the translation of HG03 open reading frame. The nucleotide sequence shown is (SEQ.ID.:1). The amino acid sequence shown is (SEQ.ID.:2).
Figure 4 shows the results of a Northern blot of the expression of HG03 mRNA in various human tissues.
Figure 5 shows the alignment of the amino acid sequence of HG03 with the amino acid sequence of the human platelet activating factor receptor (SEQ.ID.:3).
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 HG03 protein preparation that is substantially free from other proteins will contain, as a percent of ivts 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-HG03 proteins. Whether a given HG03 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 HG03 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 I%, and even more preferably no more than 0.1%, of non-HG03 nucleic acids. Whether a given HG03 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.
"Functional equivalent" means a receptor which does not have the exact same amino acid sequence o~ a naturally occurring G
protein-coupled receptor; due to alternative splicing, deletions, mutations, or additions, but retaiins at least 1%, preferably 10%, and more preferably 25% of the biological activity of the naturally occurring receptor. Such derivatives will have a significant homology with a natural GPCR and genes and cDNA encoding such. derivatives can be detected by reduced stringency hybridization with a DNA sequence encoding a natural GPCR.
The nucleic acid encoding a functional equivalent has at least about 50%
homology at the nucleotide level to a naturally occurring receptor nucleic acid.
A polypep~tide has "substantially the same biological activity" as HG03 if that polypeptide has a l~d for a ligand that is no more than 5-fold greater than the Kd of HG03 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, valine, 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).
One aspect of this invention is the identification and cloning of a novel G protein-coupled receptor (GPCR), substantially free from receptor associated proteins, designated as HG03.
Another .aspect of this invention is nucleic acids which encode the HG03 G protein-coupled receptor. These nucleic acids are substantially free from associated nucleic acids. For most cloning purposes, DNA is a preferred nucleic acid.
The present invention provides a cDNA molecule substantially free from other nucleic acids having the nucleotide sequence shown in FIGURE 1 as SEQ.ID.NO.: l . Analysis of FIGURE 3A-C revealed that SEQ.ID.NO:1 contains an open reading frame at positions 346-1419. Thus, the present invention also provides a cDNA molecule substantiially free from other nucleic acids comprising the nucleotide sequence of positions 34fi-1419 of SEQ.ID.NO.:1. The present invention also provides recombinant DrfA molecules comprising the nucleotide sequence of positions 346-1419 of SEQ.ID.NO.:1.
Sequences analysis of the open reading frame of the HG03 cDNA revealed that it encodes a protein of 358 amino acids. Based on its predicted amino acid sequence, HG03 most likely represents a novel GPCR. Northern blot analysis showed that HG03 RNA is highly expressed in the prostate, placenta, and trachea in human with a major transcript of ~1.8 kb a.nd a minor transcript of ~8.0 kb. HG03 was also expressed at lower levels in thymus and testis as a transcript of ~1.8 kb.
HG03 appears to be rE>lated to the members of receptors for nucleotides and platelet-activating factor.
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 HG03 protein preparation that is substantially free from other proteins will contain, as a percent of ivts 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-HG03 proteins. Whether a given HG03 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 HG03 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 I%, and even more preferably no more than 0.1%, of non-HG03 nucleic acids. Whether a given HG03 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.
"Functional equivalent" means a receptor which does not have the exact same amino acid sequence o~ a naturally occurring G
protein-coupled receptor; due to alternative splicing, deletions, mutations, or additions, but retaiins at least 1%, preferably 10%, and more preferably 25% of the biological activity of the naturally occurring receptor. Such derivatives will have a significant homology with a natural GPCR and genes and cDNA encoding such. derivatives can be detected by reduced stringency hybridization with a DNA sequence encoding a natural GPCR.
The nucleic acid encoding a functional equivalent has at least about 50%
homology at the nucleotide level to a naturally occurring receptor nucleic acid.
A polypep~tide has "substantially the same biological activity" as HG03 if that polypeptide has a l~d for a ligand that is no more than 5-fold greater than the Kd of HG03 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, valine, 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).
One aspect of this invention is the identification and cloning of a novel G protein-coupled receptor (GPCR), substantially free from receptor associated proteins, designated as HG03.
Another .aspect of this invention is nucleic acids which encode the HG03 G protein-coupled receptor. These nucleic acids are substantially free from associated nucleic acids. For most cloning purposes, DNA is a preferred nucleic acid.
The present invention provides a cDNA molecule substantially free from other nucleic acids having the nucleotide sequence shown in FIGURE 1 as SEQ.ID.NO.: l . Analysis of FIGURE 3A-C revealed that SEQ.ID.NO:1 contains an open reading frame at positions 346-1419. Thus, the present invention also provides a cDNA molecule substantiially free from other nucleic acids comprising the nucleotide sequence of positions 34fi-1419 of SEQ.ID.NO.:1. The present invention also provides recombinant DrfA molecules comprising the nucleotide sequence of positions 346-1419 of SEQ.ID.NO.:1.
Sequences analysis of the open reading frame of the HG03 cDNA revealed that it encodes a protein of 358 amino acids. Based on its predicted amino acid sequence, HG03 most likely represents a novel GPCR. Northern blot analysis showed that HG03 RNA is highly expressed in the prostate, placenta, and trachea in human with a major transcript of ~1.8 kb a.nd a minor transcript of ~8.0 kb. HG03 was also expressed at lower levels in thymus and testis as a transcript of ~1.8 kb.
HG03 appears to be rE>lated to the members of receptors for nucleotides and platelet-activating factor.
The novel DNA sequences of the present invention encoding HG03, in whole or in part, can be linked with other DNA sequences, i.e., DNA sequences to whiich HG03 is not naturally linked, to form "recombinant DNA molecules" containing HG03. The novel DNA
sequences of the present invention can be inserted into vectors which comprise nucleic acids encoding a GPCR or a functional equivalent. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA
vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cos:mids, yeast artificial chromosomes and other forms of episomal or integrai~ed DNA that can encode a GPCR. It is Well within the skilled artisan to eletermine an appropriate vector for a particular gene transfer or other use.
Included in the present invention are cDNA sequences that hybridize to SEQ.ID.NO.: I 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, SX Denhardt's solution, and 100 pg/ml denatured salmon sperm DNA.
Filters are hybridized for 12 to 48 hrs at 65°C in prehybridization mixture containing I00 pg/ml denatured salmion sperm DNA and 5-20 X 106 cpm of 32P-labeled probe.
Washing of filters is done at 37°C for I hr in a solution containing 2X
SSC, O.I%
SDS. This is followed by a wash in 0.1X 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 earned out in SXSSC, 5X Denhardt's solution, 50%
formamide at 42°C for 12 to 48 hours or a washing step earned 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 CloninyA Lahoratory 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 HG03. Such _b_ recombinant host cells can be cultured under suitable conditions to produce HG03.
An expression vector containing DNA encoding HG03 can be used for expression of HG03 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 S 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. CeII lines derived from mammalian species which are suitable for recombinant expression of HG03 and which are commercially available, iinclude 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 I650), 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 17I, ).
Human ennbryonic kidney (HEK 293) cells and Chinese hamster ovary (CHO) cells are particularly suitable for expression of the HG03 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 HG03 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 mammallian expression vectors can be used to express recombinant HG03 in rna~rnmalian cells. Commercially available mammalian expression vectors which are suitable include, but are not limited to, pCR2.1 (Invitrogen), pMClneo (,3tratagene), 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 (ATC(J 37198), and pSV2-dhfr (ATCC 37146). Following expression in recornbinar~t cells, HG03 can be purified by conventional techniques to a level that is substantially free from other proteins.
As with many receptor proteins, it is possible to modify many of the amino acids of HG03, 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 HG03 polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as HG03. 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 ~unino acid substitution has been made in SEQ.ID.N0.:2 wherein the polypeptides still retain substantially the same biological activity as HG03. The present invention also includes polypeptides where two or more amino acid substitutions have been made in SEQ.ID.N0.:2 wherein the polypeptides still retain substantially the same biological activity as HG03. 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 i S HG03.
The present invention also includes C-terminal truncated forms of HG03, 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 17 kd of the receptors. Accordingly, the pre sent invention includes dimers of HG03 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. Cornm. 227:200-204; Hebert et al., 1996, J. Biol. Chem. 271, 16392; Lofts et al., Onco;gene 8:281.3-2820). Accordingly, the present invention provides peptides derived from the seven membrane spanning regions of HG03 and their use to inhibit HG03 function. Such peptides can include the whole or parts of the receptor membrane spanning domains.
The present invention also includes chimer~ic HG03 proteins. Chimeric HG03 proteins consist of a contiguous polypeptide sequence of HG03 fused in frame 7_ to a polypeptide sequence of a non-HG03 protein. For example, the N-terminal domain and seven transmembrane spanning domains of HG03 fused at the C-terminus in frame to a G protein would be a chimeric HG03 protein.
The present invention also includes HG03 proteins that are in the form of multimeric structures, e.g., dimers. Such multimers of other G-protein coupled receptors are known (Helbert 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, 28612-2861ti).
The present invention also includes isolated forms of HG03 proteins.
By "isolated HG03 protein" is meant HG03 protein that has been isolated from a natural source. Use of the term "isolated" indicates that HG03 protein has been removed from its normal cellular environment. Thus, an isolated HG03 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 HG03 protein is at least 95% free of non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the HG03 protein. Thus, an HG03 protein that is expressed in bacteria or even in eukaryotic cells which do not naturally (i.e., without human interventiion) express it through recombinant means is an "isolated HG03 protein."
The specificity of binding of compounds showing affinity for HG03 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 HG03 or that inhibit the binding of a known, radiolabeled lig;and of HG03 to these cells, or membranes prepared from these cells, provides an effective method for the rapid selection of compounds with high offnity for HG03. Such ligands need not necessarily be radiolabeled but can also be nonisotopic compounds that can be used to displace bound radiolabeled compounds or that can bc~ used as activators in functional assays. Compounds identified by the above rr~ethod are likely to be agonists or antagonists of HG03 and may be peptides, proteins, or non-proteinaceous organic molecules.
Therefore, the present invention include assays by which HG03 agonists and antagonists may be identi~.ed. 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 HG03. For _g_ 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, harvesting the transfected cells and resuspending the celhe 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 lcnown agonist of the neurokinin receptor to the neurokinin receptor. :ff 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.
Accordin3aly, the present invention includes a method for determining whether .a substance is a potential agonist or antagonist of HG03 that comprises:
(a) transfecting cells with an expression vector encoding HG03;
(b) allowing the transfected cells to grow fox a time sufficient to allow HG~03 to be expressed;
(c) harvesting the transfected cells and resuspending the cells in the presence o:f a known labeled agonist of HG03 in the presence and in the absence of i;he substance;
(d) measuring the binding of the labeled agonist to HG03;
where if the amount o:f 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 HG03.
In a modification of the above-described method, step (b) is modified in that the cells are stably transfected with the expression vector containing HG03. In <~nother modification of the above-described method, step (c) is modified in ~~that the cells are not harvested and resuspended but rather the radioactive:fy labeled known agonist and the substance are _g_ .. WO 00/08133 PCT/US99/17388 contacted with the ce:Lls 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 interacaions: 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 part;icular 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-7. (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), C130-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), H~La (ATCC CCL 2), C 127I (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 ins capable of binding to HG03, i.e., whether the substance is a potential agonist; or an antagonist of HG03, where the method comprises:
(a) providing test cells by transfecting cells with an expression vector that; directs the expression of HG03 in the cells;
(b) exposing the test cells to the substance;
(c) measuring t;he amount of binding of the substance to HG03;
(d) comparing the amount of binding of the substance to HG03 in the test cells with the amount of binding of the substance to control cells that have not been transfected with HG03;
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 HG03. 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 axe typically used in the art for the study of protein-Iigand 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 HG03. Transfection is meant to include any method known in the art for introducing HG03 into the test cells. For exaimple, transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing HG03, and electroporation.
Where banding of the substance or agonist to HG03 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 particul'~ar embodiments of the above-described methods, HG03 has an amino acid sequence of SEQ.ID.~10.:2.
The above-described methods can be modified in that, rather than exposing the teslt cells to the substance, membranes can be prepared from the test cells an~i those membranes can be exposed to the substance.
Such a modification 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 HG03 comprising:
(a) providing test cells by transfecting cells with an expression vector that; directs the expression of HG03 in the cells;
(b) preparing membranes containing HG03 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 HG03 in the membranes from the test cells;
(d) comparing the amount of binding of the substance to HG03 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 HG03;
where HG03 has an amino acid sequence of SEQ:ID.N0.:2;
S where if the amount of binding of the substance to HG03 in the membranes from the test cells is greater than the amount of binding of the substance to the membranes from the control cells, then the substance is capable of binding to HG03.
The present invention provides a method for determining whether a substance is capable of binding to HG03 comprising:
(a) providing test cells by transfecting cells with an expression vector that; directs the expression of HG03 in the cells;
(b) exposing the test cells to a ligand of HG03 under conditions such that the ligand binds to the HG03 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 HG03;
(d) measuring the amount of binding of the ligand to HG03 in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HG03 in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HG03 in the presence of the substance indicates that the substance is capable of binding to HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
The present invention provides a method for determining whether a substance is capable of binding to HG03 comprising:
{a) providing test cells by transfecting cells with an expression vector that, directs the expression of HG03 in the cells;
(b) preparing membranes containing HG03 from the test cells and exposing the membranes to a ligand of HG03 under conditions such that the ligand binds to the HG03 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 HG03 in the membranes in the presence and the absence of the substance;
WO 00/08133 PCTlUS99/t7388 (e) comparing the amount of binding of the ligand to HG03 in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the iigand to HG03 in the membranes in the presence of the substance indicates that the substance is capable of binding to HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
As a further modification of the above-described methods, RNA encoding HG03 can be prepared as, e.g., by ira vitro transcription using a plasmid containing HG03 under the control of a bacteriophage T7 promoter, and the RNA can be microinjected into Xenopus oocytes in order to cause the expression of HG03 in the oocytes. Substances are then tested for binding to the HG03 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 HG03 agonists and antagonists may be identified by their ability to stimulate or antagonize a functional response mediated by HG03. HG03 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 Gj3 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 Gals or Gal6. Upon the addition of an agonist of the GPCR to the transfected cells, the GPCR was activated and was able, via Gal5 or Gal6, 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 HG03, even in S the absence of knowledge of the G-protein with which HG03 is coupled in vivo. One possibility i.s to create a fusion or chimeric protein composed of the extracellular and :membrane spanning portion of HG03 fused to a promiscuous G-protein. Such a fusion protein would be expected to transduce a signal foll',owing binding of ligand to the HG03 portion of the fusion protein. Accordingly, the present invention provides a method of identifying antagonists of HG03 comprising:
(a) providing cells that expresses a chimeric HG03 protein fused at its C-terminus to a promiscuous G-protein;
(b) exposing the cells to an agonist of HG03;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG03;
(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 HG03.
Another 3possibility for utilizing promiscuous G-proteins in connection with HGOf~ includes a method of identifying agonists of HG03 comprising:
(a) providing cells that expresses both HG03 and a promiscuous G-protein;
(b) exposing the cells to a substance that is a suspected agonist of HG03;
(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 agonist indicates that the substance is an agonist of HG03.
Levels of inositol phosphates can be measured by monitoring calcium mobilization. Intracellular calcium mobilization is typically assayed in whole cells under a xnicroscope 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 eukaryoti.c cells. In another embodiment, the cells are mammalian cells. In other embodiments, the cells axe 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), C~-1 (ATCC CCL 70), COS-1 (ATCC CRL 1F50), 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) 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 HG03 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 55°C.
In a partiicular embodiment of the above-described method, the promiscuous G-protein is selected from the group consisting of Gals or Gal6. Expression vectors containing Gals or Gal6 are known in the art.
See, e.g., Offermanns; Buhl et cal., 1993, FEBS Lett. 323:132-134;
Amatruda et al., 1993, J. Biol. Chem. 268:10139-10144.
The above-described assay can be easily modified to form a method to identify antagonists of HG03. Such a method is also part of the present invention and comprises:
(a) providing cells that expresses both HG03 and a proxniscuaus G-proteixx;
(b) exposing the cells to a substance that is an agonist of HG03;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG03;
(d) measuring the level of inositol phosphates in the cells;
_ 15-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 the cells in the absence of the suspected antagonist indicates that the substance is an antagonist of HG03.
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 15'73), Raji (ATCC CCL 86), C~-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL /651), C~iO-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).
The conditions under which steps (b) and (c) of the method are 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 transfected with expression vectors that direct the expression of HG03 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 Gal5 or Gal6.
In particular embodiments of the above-described methods, HG03 has an amino acid sequence of SEQ.ID.Io10.:2.
Another embodiment of the present invention are methods of expressing HG03 in recombinant systems and of identifying agonists and antagonists of HG03, and for counter-screening.
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 axe 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 t:he 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, $iolTechnology 10:973-980, at 980). HG03 proteins and DNA encoding HG03 proteins have utility in that they can be used as "minus targets" in screens design to identify compounds that specifically interact wiith other G-protein coupled receptors.
The present invention also includes antibodies to the HG03 protein. Such antibodies may be polyclonal antibodies or monoclonal antibodies. The antibodies of the present invention are raised against the entire HG03 protein o:r 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, HG03 protein or an antigenic fragment, coupled to a suitable corner, 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 fox the presence of antibodies to the injected antigen. The injections can be intrarnuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant.
For the production of monoclonal antibodies, HG03 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 anima:( is generally a mouse. The animal's spleen cells are then immortalized, ofl:en 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, sere Antibodies: A Laboratory Manual, Harlow & Lane, eds., Cold Spring Harbor Laboratory Press, 1988.
Gene therapy may be used to introduce HG03 polypeptides into the cells of target organs. Nucleotides encoding HG03 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, andl polio virus based vectors. Alternatively, nucleotides encoding HG03 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 arcs suitable for ex vivo as well as in vivo gene therapy. Gene therapy with HG03 polypeptides will be particularly useful for the treatment of diseases where it is beneficial to elevate HG03 activity.
The following non-limiting examples are presented to better illustrate the invention.
Cloning and seauencin~of HG03 The full-length coding sequence of HG03 was isolated by multiple rounds of RCCA from a prostate cDNA library. Originally, the HG03 gene was named AOMF15 but wa;> designated later HG03. The primer pair, F216 +R369 was initially used to scan the 1-2.SKB and 2.SKB prostate cDNA libraries. Positive pools were identified where nested insert-'vector PCRs were then carried out using the following combinations of primers: first round PCR reactions, F216+5438, F216+873F, and 8369+5~43R, 8369+873F; nested second round PCR reaction, F325+5788, 8280+383F'. The list of primers used for the isolation of HG03:
AOMF15.F216 CC'.AGGCAAACATTACACGCA (SEQ.ID.N0.:4) AOMF15.F325 GF~ATTCTGCACAAGTGATACGGTA (SEQ.ID.N0.:5) AOMF15.R280 CP~GGCTTTTAGATGAATCTGCA (SEQ.ID.NO.:C) AOMF15.R369 ACPCATCAGGGTTGTTGTGGC (SEQ.ID.N0.:7) HG03.37R GC',GTGTCAGC~AAACACTTGG (SEQ.ID.N0.:8) HG03 FL243F AF~AGAAATCAAACCAGGAATAACC (SQ.ID.N0.:9) HG03 FL1429R CTTTGTACATATCGATTCCAACACAC (SEQ.ID.NO.:10) _ x$ _ PBS.873F CCCAGGCTTTACACTTTATGCTTCC (SEQ.ID.N0.:11) PBS.543R GGGGATGTGC'TGCAAGGCGA (SEQ.ID.N0.:12) PCR reactions were corned out with AmpliTaq (Perkin Elmer, CA and Taq extender (Stratagene) under the conditions suggested by the supplier of Taq extender. The PCR fragments were sequenced and assembled. Complete sequence was obtained by using primers 378 and 11188 by primer walking.
The analysis of this "race" PCR and sequencing reactions resulted in the assembly of a contiguous fragment of 1478 base pairs. This sequence contains an open reading frame of 35.'3 amino acids. The full length coding sequence of was amplified by using primers FL-243F and FL-14298 and cloned into the plasmid vector pCR2.1 by TA cloning (Invitrogen, Carlsbad, CA). It is well within the skilled artisan to determine an appropriate vector for a particular gene transfer or other use.
:EXAMPLE 2 Tissue distribution of HG03 RNA transcripts An approximately 1.8 kb fragment containing HG03 was randomly labelled with 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 HG03 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 autoradiography. The results are shown in Figure 4.
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.
sequences of the present invention can be inserted into vectors which comprise nucleic acids encoding a GPCR or a functional equivalent. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA
vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cos:mids, yeast artificial chromosomes and other forms of episomal or integrai~ed DNA that can encode a GPCR. It is Well within the skilled artisan to eletermine an appropriate vector for a particular gene transfer or other use.
Included in the present invention are cDNA sequences that hybridize to SEQ.ID.NO.: I 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, SX Denhardt's solution, and 100 pg/ml denatured salmon sperm DNA.
Filters are hybridized for 12 to 48 hrs at 65°C in prehybridization mixture containing I00 pg/ml denatured salmion sperm DNA and 5-20 X 106 cpm of 32P-labeled probe.
Washing of filters is done at 37°C for I hr in a solution containing 2X
SSC, O.I%
SDS. This is followed by a wash in 0.1X 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 earned out in SXSSC, 5X Denhardt's solution, 50%
formamide at 42°C for 12 to 48 hours or a washing step earned 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 CloninyA Lahoratory 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 HG03. Such _b_ recombinant host cells can be cultured under suitable conditions to produce HG03.
An expression vector containing DNA encoding HG03 can be used for expression of HG03 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 S 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. CeII lines derived from mammalian species which are suitable for recombinant expression of HG03 and which are commercially available, iinclude 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 I650), 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 17I, ).
Human ennbryonic kidney (HEK 293) cells and Chinese hamster ovary (CHO) cells are particularly suitable for expression of the HG03 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 HG03 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 mammallian expression vectors can be used to express recombinant HG03 in rna~rnmalian cells. Commercially available mammalian expression vectors which are suitable include, but are not limited to, pCR2.1 (Invitrogen), pMClneo (,3tratagene), 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 (ATC(J 37198), and pSV2-dhfr (ATCC 37146). Following expression in recornbinar~t cells, HG03 can be purified by conventional techniques to a level that is substantially free from other proteins.
As with many receptor proteins, it is possible to modify many of the amino acids of HG03, 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 HG03 polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as HG03. 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 ~unino acid substitution has been made in SEQ.ID.N0.:2 wherein the polypeptides still retain substantially the same biological activity as HG03. The present invention also includes polypeptides where two or more amino acid substitutions have been made in SEQ.ID.N0.:2 wherein the polypeptides still retain substantially the same biological activity as HG03. 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 i S HG03.
The present invention also includes C-terminal truncated forms of HG03, 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 17 kd of the receptors. Accordingly, the pre sent invention includes dimers of HG03 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. Cornm. 227:200-204; Hebert et al., 1996, J. Biol. Chem. 271, 16392; Lofts et al., Onco;gene 8:281.3-2820). Accordingly, the present invention provides peptides derived from the seven membrane spanning regions of HG03 and their use to inhibit HG03 function. Such peptides can include the whole or parts of the receptor membrane spanning domains.
The present invention also includes chimer~ic HG03 proteins. Chimeric HG03 proteins consist of a contiguous polypeptide sequence of HG03 fused in frame 7_ to a polypeptide sequence of a non-HG03 protein. For example, the N-terminal domain and seven transmembrane spanning domains of HG03 fused at the C-terminus in frame to a G protein would be a chimeric HG03 protein.
The present invention also includes HG03 proteins that are in the form of multimeric structures, e.g., dimers. Such multimers of other G-protein coupled receptors are known (Helbert 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, 28612-2861ti).
The present invention also includes isolated forms of HG03 proteins.
By "isolated HG03 protein" is meant HG03 protein that has been isolated from a natural source. Use of the term "isolated" indicates that HG03 protein has been removed from its normal cellular environment. Thus, an isolated HG03 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 HG03 protein is at least 95% free of non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the HG03 protein. Thus, an HG03 protein that is expressed in bacteria or even in eukaryotic cells which do not naturally (i.e., without human interventiion) express it through recombinant means is an "isolated HG03 protein."
The specificity of binding of compounds showing affinity for HG03 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 HG03 or that inhibit the binding of a known, radiolabeled lig;and of HG03 to these cells, or membranes prepared from these cells, provides an effective method for the rapid selection of compounds with high offnity for HG03. Such ligands need not necessarily be radiolabeled but can also be nonisotopic compounds that can be used to displace bound radiolabeled compounds or that can bc~ used as activators in functional assays. Compounds identified by the above rr~ethod are likely to be agonists or antagonists of HG03 and may be peptides, proteins, or non-proteinaceous organic molecules.
Therefore, the present invention include assays by which HG03 agonists and antagonists may be identi~.ed. 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 HG03. For _g_ 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, harvesting the transfected cells and resuspending the celhe 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 lcnown agonist of the neurokinin receptor to the neurokinin receptor. :ff 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.
Accordin3aly, the present invention includes a method for determining whether .a substance is a potential agonist or antagonist of HG03 that comprises:
(a) transfecting cells with an expression vector encoding HG03;
(b) allowing the transfected cells to grow fox a time sufficient to allow HG~03 to be expressed;
(c) harvesting the transfected cells and resuspending the cells in the presence o:f a known labeled agonist of HG03 in the presence and in the absence of i;he substance;
(d) measuring the binding of the labeled agonist to HG03;
where if the amount o:f 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 HG03.
In a modification of the above-described method, step (b) is modified in that the cells are stably transfected with the expression vector containing HG03. In <~nother modification of the above-described method, step (c) is modified in ~~that the cells are not harvested and resuspended but rather the radioactive:fy labeled known agonist and the substance are _g_ .. WO 00/08133 PCT/US99/17388 contacted with the ce:Lls 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 interacaions: 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 part;icular 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-7. (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), C130-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), H~La (ATCC CCL 2), C 127I (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 ins capable of binding to HG03, i.e., whether the substance is a potential agonist; or an antagonist of HG03, where the method comprises:
(a) providing test cells by transfecting cells with an expression vector that; directs the expression of HG03 in the cells;
(b) exposing the test cells to the substance;
(c) measuring t;he amount of binding of the substance to HG03;
(d) comparing the amount of binding of the substance to HG03 in the test cells with the amount of binding of the substance to control cells that have not been transfected with HG03;
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 HG03. 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 axe typically used in the art for the study of protein-Iigand 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 HG03. Transfection is meant to include any method known in the art for introducing HG03 into the test cells. For exaimple, transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing HG03, and electroporation.
Where banding of the substance or agonist to HG03 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 particul'~ar embodiments of the above-described methods, HG03 has an amino acid sequence of SEQ.ID.~10.:2.
The above-described methods can be modified in that, rather than exposing the teslt cells to the substance, membranes can be prepared from the test cells an~i those membranes can be exposed to the substance.
Such a modification 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 HG03 comprising:
(a) providing test cells by transfecting cells with an expression vector that; directs the expression of HG03 in the cells;
(b) preparing membranes containing HG03 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 HG03 in the membranes from the test cells;
(d) comparing the amount of binding of the substance to HG03 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 HG03;
where HG03 has an amino acid sequence of SEQ:ID.N0.:2;
S where if the amount of binding of the substance to HG03 in the membranes from the test cells is greater than the amount of binding of the substance to the membranes from the control cells, then the substance is capable of binding to HG03.
The present invention provides a method for determining whether a substance is capable of binding to HG03 comprising:
(a) providing test cells by transfecting cells with an expression vector that; directs the expression of HG03 in the cells;
(b) exposing the test cells to a ligand of HG03 under conditions such that the ligand binds to the HG03 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 HG03;
(d) measuring the amount of binding of the ligand to HG03 in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HG03 in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HG03 in the presence of the substance indicates that the substance is capable of binding to HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
The present invention provides a method for determining whether a substance is capable of binding to HG03 comprising:
{a) providing test cells by transfecting cells with an expression vector that, directs the expression of HG03 in the cells;
(b) preparing membranes containing HG03 from the test cells and exposing the membranes to a ligand of HG03 under conditions such that the ligand binds to the HG03 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 HG03 in the membranes in the presence and the absence of the substance;
WO 00/08133 PCTlUS99/t7388 (e) comparing the amount of binding of the ligand to HG03 in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the iigand to HG03 in the membranes in the presence of the substance indicates that the substance is capable of binding to HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
As a further modification of the above-described methods, RNA encoding HG03 can be prepared as, e.g., by ira vitro transcription using a plasmid containing HG03 under the control of a bacteriophage T7 promoter, and the RNA can be microinjected into Xenopus oocytes in order to cause the expression of HG03 in the oocytes. Substances are then tested for binding to the HG03 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 HG03 agonists and antagonists may be identified by their ability to stimulate or antagonize a functional response mediated by HG03. HG03 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 Gj3 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 Gals or Gal6. Upon the addition of an agonist of the GPCR to the transfected cells, the GPCR was activated and was able, via Gal5 or Gal6, 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 HG03, even in S the absence of knowledge of the G-protein with which HG03 is coupled in vivo. One possibility i.s to create a fusion or chimeric protein composed of the extracellular and :membrane spanning portion of HG03 fused to a promiscuous G-protein. Such a fusion protein would be expected to transduce a signal foll',owing binding of ligand to the HG03 portion of the fusion protein. Accordingly, the present invention provides a method of identifying antagonists of HG03 comprising:
(a) providing cells that expresses a chimeric HG03 protein fused at its C-terminus to a promiscuous G-protein;
(b) exposing the cells to an agonist of HG03;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG03;
(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 HG03.
Another 3possibility for utilizing promiscuous G-proteins in connection with HGOf~ includes a method of identifying agonists of HG03 comprising:
(a) providing cells that expresses both HG03 and a promiscuous G-protein;
(b) exposing the cells to a substance that is a suspected agonist of HG03;
(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 agonist indicates that the substance is an agonist of HG03.
Levels of inositol phosphates can be measured by monitoring calcium mobilization. Intracellular calcium mobilization is typically assayed in whole cells under a xnicroscope 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 eukaryoti.c cells. In another embodiment, the cells are mammalian cells. In other embodiments, the cells axe 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), C~-1 (ATCC CCL 70), COS-1 (ATCC CRL 1F50), 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) 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 HG03 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 55°C.
In a partiicular embodiment of the above-described method, the promiscuous G-protein is selected from the group consisting of Gals or Gal6. Expression vectors containing Gals or Gal6 are known in the art.
See, e.g., Offermanns; Buhl et cal., 1993, FEBS Lett. 323:132-134;
Amatruda et al., 1993, J. Biol. Chem. 268:10139-10144.
The above-described assay can be easily modified to form a method to identify antagonists of HG03. Such a method is also part of the present invention and comprises:
(a) providing cells that expresses both HG03 and a proxniscuaus G-proteixx;
(b) exposing the cells to a substance that is an agonist of HG03;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG03;
(d) measuring the level of inositol phosphates in the cells;
_ 15-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 the cells in the absence of the suspected antagonist indicates that the substance is an antagonist of HG03.
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 15'73), Raji (ATCC CCL 86), C~-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL /651), C~iO-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).
The conditions under which steps (b) and (c) of the method are 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 transfected with expression vectors that direct the expression of HG03 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 Gal5 or Gal6.
In particular embodiments of the above-described methods, HG03 has an amino acid sequence of SEQ.ID.Io10.:2.
Another embodiment of the present invention are methods of expressing HG03 in recombinant systems and of identifying agonists and antagonists of HG03, and for counter-screening.
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 axe 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 t:he 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, $iolTechnology 10:973-980, at 980). HG03 proteins and DNA encoding HG03 proteins have utility in that they can be used as "minus targets" in screens design to identify compounds that specifically interact wiith other G-protein coupled receptors.
The present invention also includes antibodies to the HG03 protein. Such antibodies may be polyclonal antibodies or monoclonal antibodies. The antibodies of the present invention are raised against the entire HG03 protein o:r 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, HG03 protein or an antigenic fragment, coupled to a suitable corner, 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 fox the presence of antibodies to the injected antigen. The injections can be intrarnuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant.
For the production of monoclonal antibodies, HG03 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 anima:( is generally a mouse. The animal's spleen cells are then immortalized, ofl:en 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, sere Antibodies: A Laboratory Manual, Harlow & Lane, eds., Cold Spring Harbor Laboratory Press, 1988.
Gene therapy may be used to introduce HG03 polypeptides into the cells of target organs. Nucleotides encoding HG03 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, andl polio virus based vectors. Alternatively, nucleotides encoding HG03 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 arcs suitable for ex vivo as well as in vivo gene therapy. Gene therapy with HG03 polypeptides will be particularly useful for the treatment of diseases where it is beneficial to elevate HG03 activity.
The following non-limiting examples are presented to better illustrate the invention.
Cloning and seauencin~of HG03 The full-length coding sequence of HG03 was isolated by multiple rounds of RCCA from a prostate cDNA library. Originally, the HG03 gene was named AOMF15 but wa;> designated later HG03. The primer pair, F216 +R369 was initially used to scan the 1-2.SKB and 2.SKB prostate cDNA libraries. Positive pools were identified where nested insert-'vector PCRs were then carried out using the following combinations of primers: first round PCR reactions, F216+5438, F216+873F, and 8369+5~43R, 8369+873F; nested second round PCR reaction, F325+5788, 8280+383F'. The list of primers used for the isolation of HG03:
AOMF15.F216 CC'.AGGCAAACATTACACGCA (SEQ.ID.N0.:4) AOMF15.F325 GF~ATTCTGCACAAGTGATACGGTA (SEQ.ID.N0.:5) AOMF15.R280 CP~GGCTTTTAGATGAATCTGCA (SEQ.ID.NO.:C) AOMF15.R369 ACPCATCAGGGTTGTTGTGGC (SEQ.ID.N0.:7) HG03.37R GC',GTGTCAGC~AAACACTTGG (SEQ.ID.N0.:8) HG03 FL243F AF~AGAAATCAAACCAGGAATAACC (SQ.ID.N0.:9) HG03 FL1429R CTTTGTACATATCGATTCCAACACAC (SEQ.ID.NO.:10) _ x$ _ PBS.873F CCCAGGCTTTACACTTTATGCTTCC (SEQ.ID.N0.:11) PBS.543R GGGGATGTGC'TGCAAGGCGA (SEQ.ID.N0.:12) PCR reactions were corned out with AmpliTaq (Perkin Elmer, CA and Taq extender (Stratagene) under the conditions suggested by the supplier of Taq extender. The PCR fragments were sequenced and assembled. Complete sequence was obtained by using primers 378 and 11188 by primer walking.
The analysis of this "race" PCR and sequencing reactions resulted in the assembly of a contiguous fragment of 1478 base pairs. This sequence contains an open reading frame of 35.'3 amino acids. The full length coding sequence of was amplified by using primers FL-243F and FL-14298 and cloned into the plasmid vector pCR2.1 by TA cloning (Invitrogen, Carlsbad, CA). It is well within the skilled artisan to determine an appropriate vector for a particular gene transfer or other use.
:EXAMPLE 2 Tissue distribution of HG03 RNA transcripts An approximately 1.8 kb fragment containing HG03 was randomly labelled with 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 HG03 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 autoradiography. The results are shown in Figure 4.
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 (20)
1. A recombinant DNA molecule encoding a polypeptide having an amino acid sequence of SEQ.ID.NO.:2.
2. The DNA molecule of Claim 1 comprising a nucleotide sequence selected from the group consisting of SEQ.ID.NO.:1 and positions 346-of SEQ.ID.NO.:1.
3. A DNA molecule that hybridizes under stringent conditions to the DNA of Claim 1.
4. An expression vector comprising the DNA of Claim 1.
5. A recombinant host cell comprising the DNA of Claim 1.
6. An HG03 protein, substantially free from other proteins, having an amino acid sequence of SEQ.ID.N0.:2.
7. The HG03 protein of Claim 6 containing a single amino acid substitution.
8. The HG03 protein of Claim 7 where the substitution is a conservative substitution.
9. The HG03 protein of Claim 6 containing two or more amino acid substitutions and substantially the same biological activity as HG03.
10. A method for determining whether a substance is capable of binding to HG03 comprising:
(a) providing test cells by transfecting cells with an expression vector than directs the expression of HG03 in the cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of binding of the substance to HG03;
(d) comparing the amount of binding of the substance to HG03 in the test cells with the amount of binding of the substance to control cells that have not been transfected with HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2;
where if the amount of binding of the substance to HG03 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 HG03.
(a) providing test cells by transfecting cells with an expression vector than directs the expression of HG03 in the cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of binding of the substance to HG03;
(d) comparing the amount of binding of the substance to HG03 in the test cells with the amount of binding of the substance to control cells that have not been transfected with HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2;
where if the amount of binding of the substance to HG03 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 HG03.
11. A method for determining whether a substance is capable of binding to HG03 comprising:
(a) providing test cells by transfecting cells with an expression vector than directs the expression of HG03 in the cells;
(b) preparing membranes containing HG03 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 HG03 in the membranes from the test cells;
(d) comparing the amount of binding of the substance to HG03 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 HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2;
where if the amount of binding of the substance to HG03 in the membranes from the test cells is greater than the amount of binding of the substance to the membranes from the control cells, then the substance is capable of binding to HG03.
(a) providing test cells by transfecting cells with an expression vector than directs the expression of HG03 in the cells;
(b) preparing membranes containing HG03 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 HG03 in the membranes from the test cells;
(d) comparing the amount of binding of the substance to HG03 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 HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2;
where if the amount of binding of the substance to HG03 in the membranes from the test cells is greater than the amount of binding of the substance to the membranes from the control cells, then the substance is capable of binding to HG03.
12. A method for determining whether a substance is capable of binding to HG03 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG03 in the cells;
(b) exposing the test cells to a ligand of HG03 under conditions such that the ligand binds to the HG03 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 HG03;
(d) measuring the amount of binding of the ligand to HG03 in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HG03 in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HG03 in the presence of the substance indicates that the substance is capable of binding to HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG03 in the cells;
(b) exposing the test cells to a ligand of HG03 under conditions such that the ligand binds to the HG03 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 HG03;
(d) measuring the amount of binding of the ligand to HG03 in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HG03 in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HG03 in the presence of the substance indicates that the substance is capable of binding to HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
13. A method for determining whether a substance is capable of binding to HG03 comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG03 in the cells;
(b) preparing membranes containing HG03 from the test cells and exposing the membranes to a ligand of HG03 under conditions such that the ligand binds to the HG03 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 HG03 in the membranes in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HG03 in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HG03 in the membranes in the presence of the substance indicates that the substance is capable of binding to HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
(a) providing test cells by transfecting cells with an expression vector that directs the expression of HG03 in the cells;
(b) preparing membranes containing HG03 from the test cells and exposing the membranes to a ligand of HG03 under conditions such that the ligand binds to the HG03 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 HG03 in the membranes in the presence and the absence of the substance;
(e) comparing the amount of binding of the ligand to HG03 in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to HG03 in the membranes in the presence of the substance indicates that the substance is capable of binding to HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
14. A method for determining whether a substance is a potential agonist or antagonist of HG03 that comprises:
(a) transfecting cells with an expression vector containing HG03;
(b) allowing the transfected cells to grow for a time sufficient to allow HG03 to be expressed;
(c) harvesting the transfected cells and resuspending the cells in assay buffer containing a known labeled agonist of HG03 in the presence and in the absence of the substance;
(d) measuring the binding of the known labeled agonist to HG03;
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 HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
(a) transfecting cells with an expression vector containing HG03;
(b) allowing the transfected cells to grow for a time sufficient to allow HG03 to be expressed;
(c) harvesting the transfected cells and resuspending the cells in assay buffer containing a known labeled agonist of HG03 in the presence and in the absence of the substance;
(d) measuring the binding of the known labeled agonist to HG03;
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 HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
15. A method of identifying agonists of HG03 comprising:
(a) providing cells that expresses both HG03 and a promiscuous G-protein;
(b) exposing the cells to a substance that is a suspected agonist of HG03;
(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 HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
(a) providing cells that expresses both HG03 and a promiscuous G-protein;
(b) exposing the cells to a substance that is a suspected agonist of HG03;
(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 HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
16. A method identifying antagonists of HG03 comprising:
(a) providing cells that expresses both HG03 and a promiscuous G-protein;
(b) exposing the cells to an agonist of HG03;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG03;
(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 HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
(a) providing cells that expresses both HG03 and a promiscuous G-protein;
(b) exposing the cells to an agonist of HG03;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG03;
(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 HG03;
where HG03 has an amino acid sequence of SEQ.ID.N0.:2.
17. A method of identifying antagonists of HG03 comprising:
(a) providing cells that expresses a chimeric HG03 protein fused at its C-terminus to a promiscuous G-protein;
(b) exposing the cells to an agonist of HG03;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG03;
(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 HG03;
where the HG03 protein has an amino acid sequence of SEQ.ID.N0.:2.
(a) providing cells that expresses a chimeric HG03 protein fused at its C-terminus to a promiscuous G-protein;
(b) exposing the cells to an agonist of HG03;
(c) subsequently or concurrently to step (b), exposing the cells to a substance that is a suspected antagonist of HG03;
(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 HG03;
where the HG03 protein has an amino acid sequence of SEQ.ID.N0.:2.
18. An antibody that binds specifically to HG03 where HG03 has an amino acid sequence of SEQ.ID.N0.:2. I
19. A method of expressing a truncated version of HG03 protein comprising:
(a) transfecting a host cell with a expression vector that encodes an HG03 protein that has been truncated at the amino terminus;
(b) culturing the transfected cells of step (a) under conditions such that the truncated HG03 protein is expressed.
(a) transfecting a host cell with a expression vector that encodes an HG03 protein that has been truncated at the amino terminus;
(b) culturing the transfected cells of step (a) under conditions such that the truncated HG03 protein is expressed.
20. The method of Claim 19 where the truncated HG03 protein is a chimeric HG03 protein.
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| US60/095,571 | 1998-08-06 | ||
| PCT/US1999/017388 WO2000008133A1 (en) | 1998-08-06 | 1999-08-02 | NOVEL G PROTEIN-COUPLED RECEPTOR cDNA SEQUENCE |
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| JP (1) | JP2002526036A (en) |
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| AU6181100A (en) * | 1999-07-29 | 2001-02-19 | Helix Research Institute | Gene encoding novel serine protease-like protein |
| EP1232265A2 (en) * | 1999-11-26 | 2002-08-21 | MERCK PATENT GmbH | G-protein coupled receptor htogh35 |
| JP2003517154A (en) * | 1999-12-16 | 2003-05-20 | ミレニアム・ファーマシューティカルズ・インコーポレイテッド | 2871 receptor, G protein-coupled receptor and method of using the same |
| EP1273659A4 (en) * | 2000-04-12 | 2004-04-21 | Takeda Chemical Industries Ltd | NOVEL G PROTEIN-COUPLED RECEPTOR PROTEIN AND DNA THEREOF |
| EP1363655A4 (en) * | 2000-12-18 | 2005-04-06 | Merck & Co Inc | ISOLATED NUCLEIC ACID MOLECULES ENCODING A HUMAN G-GPR54 PROTEIN-COUPLED RECEPTOR AND MURINE, ENCODED PROTEINS, CELLS TRANSFORMED THEREFROM, AND USES THEREOF |
| AU2002217455A1 (en) * | 2000-12-20 | 2002-07-01 | Takeda Chemical Industries Ltd. | Novel g protein-coupled receptor protein and dna thereof |
| EP1392724A2 (en) * | 2001-05-16 | 2004-03-03 | Paradigm Therapeutics Limited | G-protein coupled receptor |
| CN114766546B (en) * | 2021-12-29 | 2024-03-26 | 淮阴工学院 | Application of bacillus licheniformis HG03 in preventing and controlling post-harvest soft rot of juicy peach caused by rhizopus |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4985352A (en) * | 1988-02-29 | 1991-01-15 | The Trustees Of Columbia University In The City Of New York | DNA encoding serotonin 1C (5HT1c) receptor, isolated 5HT1c receptor, mammalian cells expressing same and uses thereof |
| US5144007A (en) * | 1988-11-03 | 1992-09-01 | La Jolla Cancer Research Foundation | Thyroid hormone receptor |
-
1999
- 1999-08-02 EP EP99941986A patent/EP1105465A1/en not_active Withdrawn
- 1999-08-02 JP JP2000563760A patent/JP2002526036A/en active Pending
- 1999-08-02 CA CA002339347A patent/CA2339347A1/en not_active Abandoned
- 1999-08-02 WO PCT/US1999/017388 patent/WO2000008133A1/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| JP2002526036A (en) | 2002-08-20 |
| EP1105465A1 (en) | 2001-06-13 |
| WO2000008133A9 (en) | 2001-12-20 |
| WO2000008133A1 (en) | 2000-02-17 |
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