Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 

 

 

Title:  Identification of a G protein-coupled receptor transcriptionally regulated by protein tyrosine kinase signaling in hematopoietic cell

United States Patent:  6,569,995

Issued:  May 27, 2003

Inventors:  Weng; Zhigang (Los Angeles, CA); Witte; Owen N. (Sherman Oaks, CA)

Assignee:  The Regents of the University of California (Oakland, CA)

Appl. No.:  768670

Filed:  January 23, 2001

 

Abstract

A G protein-coupled receptor (GPCR) which is activated by oncogenes. The receptor is found predominantly in hematopoietic cells and tissues and functions as a tumor suppressor gene and induces cell cycle arrest. This receptor may play an important role in regulating the proliferation and differentiation of hematopoietic cells. Regulation of receptor activity has several therapeutic applications.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes the identification and sequencing of a novel G protein-coupled receptor (GPCR) that is transcriptionally upregulated by protein tyrosine kinase signaling and during lymphocyte activation. The GPCR functions as a tumor suppressor gene, induces cell cycle arrest during mitosis and is found on human chromosome 14q32.3, a region frequently found altered in human cancers. This GPCR was identified while studying cellular genes that can be regulated by BCR-ABL, a chimeric tyrosine kinase oncogene associated with the pathogenesis of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL) (Kurzrock, N. Engl. J. Med. 319: 990-998, 1988). Using a PCR-based differential screening technique (representational difference analysis or RDA) (Lisitsyn et al. Science 259:946-951, 1993; Hubank et al., Nucl. Acids Res. 22:5640-5648, 1994), genes expressed in murine bone marrow cells transformed by the wild type (WT) BCR-ABL were compared to those expressed when a transformation-defective mutant variant carrying a mutation in the SH2 domain of BCR-ABL was used to infect these cells. One of these differentially expressed murine genes (N2A) was predominantly expressed in hematopoietic tissues such as spleen and thymus. The cDNA and deduced amino acid sequences of the murine GPCR are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The human homologue of the mouse protein was then isolated using the murine cDNA as a probe. The corresponding human cDNA and deduced amino acid sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The GPCR protein sequence of the invention has the sequence shown in SEQ ID NOS; 2 and 4, or sequence variations thereof which do not substantially compromise the ability of these genes to be regulated by protein tyrosine kinases or sequence variations thereof which do not substantially compromise the functional activities of these proteins. It will be appreciated that GPCR proteins containing one or more amino acid replacements in various positions of the sequences shown in SEQ ID NOS: 2 and 4 are also within the scope of the invention.

Many amino acid substitutions can be made to the native sequence without compromising its functional activity. Variations of these protein sequences contemplated for use in the present invention include minor insertions, deletions and substitutions. For example, conservative amino acid replacements are contemplated. Such replacements are, for example, those that take place within a family of amino acids that are related in the chemical nature of their side chains. The families of amino acids include the basic amino acids (lysine, arginine, histidine); the acidic amino acids (aspartic acid, glutamic acid); the non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and the uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) and the aromatic amino acids (phenylalanine, tryptophan, tyrosine). In particular, it is generally accepted that conservative amino acid replacements consisting of an isolated replacement of a leucine with an isoleucine or valine, or an aspartic acid with a glutamic acid, or a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, in an area outside of the polypeptide's active site, will not have a major effect on the properties of the polypeptide.

The murine protein was determined to be a member of the GPCR superfamily by its homology to other GPCRs, including the mouse TDAG8 protein and the P2Y purinoceptor, using sequence alignment programs. The human GPCR homologue was isolated by screening a human spleen cDNA library under high stringency conditions (2.times.SSC, 0.1% SDS, 65oC.). The murine and human GPCRs share approximately seventy percent identity at the amino acid level. Any DNA molecule capable of hybridizing the DNA sequence shown in SEQ ID NO: 1 under these conditions or lower stringency conditions, as well as the protein encoded by such a DNA molecule, is within the scope of the invention.

Northern analysis of various murine tissue samples detected two GPCR transcripts of about 3 kb and 5 kb in spleen, thymus, lung and heart, but not in normal bone marrow, brain, liver, skeletal muscle or kidney. Northern analysis of human tissues showed that the human GPCR is exclusively expressed in spleen and peripheral leukocytes, but not in heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, thymus, prostate, testis, ovary, small intestine and the mucosal lining of the colon. This further suggests a role of this gene in hematopoiesis. The human GPCR is transcriptionally activated in B cells upon activation by either phorbol 12-myristate 13-acetate (PMA) plus ionomycin or anti-IgM antibodies. The activation of GPCR transcription was also observed in B cells upon irradiation with x-rays or activation by the CD40 ligand. The human GPCR transcript is also present in the ALL-1 and K-562 leukemia cell lines.

The GPCR was transcriptionally activated by BCR-ABL and v-Abl, a protein tyrosine kinase oncogene found in Abelson Murine Leukemia Virus. To our knowledge, this is the first demonstration that a GPCR can be transcriptionally regulated by a protein tyrosine kinase. Interestingly, a mutant form of BCR-ABL (carrying a mutation in the SH2 domain) that lacks oncogenic potential failed to transcriptionally activate the GPCR. In addition, Cyclin D1, an important cell cycle regulator (Afar et al., Proc. Natl. Acad. Sci. U.S.A. 92:9540-9544, 1995) that can complement the BCR-ABL mutant for transformation, restored the expression of the GPCR These data suggest that this GPCR may also be a marker for transformation by BCR-ABL and other tyrosine kinase signaling pathways.

The domains of BCR-ABL, TEL-ABL (an oncogenic fusion protein associated with leukemia) and v-ABL. TEL-ABL, Grb-2 (an adaptor protein which couples BCR-ABL to Ras) mutant and autophosphorylation mutant did not activate the GPCR. The GPCR receptor was also transcriptionally activated by v-Mos, a serine kinase oncogene that activates MAP kinase (Davis, Mol. Reprod. Dev. 42:459-67, 1995). Since v-Mos, BCR-ABL and v-ABL all activate MAP kinases (Davis, supra.), the GPCR may be directly regulated by MAP kinase signaling pathways. Therefore, it is contemplated that the GPCR may also be activated by a wide-variety of protein kinases as well as their regulators and effectors during cell growth and differentiation such as Ras, Myc, Fos, Jun and BTK.

The GPCR is expressed in spleen and thymus, but not in normal bone marrow cells, suggesting that it may play an important role in mid- and late stages of T and B cell development. During development, self-reactive immature thymocytes are clonally deleted in the thymus, a phenomenon which establishes T cell tolerance (negative selection). It has been shown that the deletion of self-reactive immature T cells in the thymus is mediated by apoptosis upon T cell receptor engagement. TDAG8, a GPCR family member, is induced in T cells during apoptosis upon T cell receptor activation (Choi et al., Cell. Immunol., 168:78-84, 1996). This suggests that TDAG8 may play a role in negative selection of T cells. Since the GPCRs that we isolated share about 30% homology with TDAG8, it is conceivable that the GPCRs may also play a role in negative selection of T cells. Sequence analysis of the GPCR with its family members reveal that they also share significant homology with the P2Y receptor, a GPCR for ATP. It has been. shown recently that P2Y receptor is transcriptionally upregulated during T cell activation (Koshiba et al., Proc. Natl. Acad. Sci. U.S.A., 94:831-836, 1997).

The GPCRs may play a role in directing migration of lymphocytes into specific anatomical compartments of spleen and thymus for maturation. Previous studies on a hematopoietic-specific GPCR, BLR1, suggest that BLR1 plays an important role for directing migration of lymphocytes into splenic follicles as well as migration of activated B cells into B cell-follicles of the spleen, a prerequisite for the development of an antigen-specific immune response (Forster et al., Cell, 87:1037-1047, 1996). Expression of GPCRs in hematopoietic-specific tissues suggest that it may also play similar roles in directing migration of lymphocytes into lymphoid organs for their maturation.

The human and murine GPCRs share about 70% identity at the amino acid level based on the translation of their complete cDNA sequences. Both the mouse and human GPCR cDNA clones can be used for in situ analysis to examine whether the expression of the receptor is restricted to certain anatomical regions of the spleen and thymus. The mouse and human genomic clones encoding the full length GPCRs were also isolated. The mouse genomic clone has been used for constructing a targeting vector to knock-out the GPCR in mice by homologous recombination. The GPCR -/- mice will allow further evaluation of the physiological functions of this receptor. The GPCR -/- mice will also allow determination of whether in vivo leukemogenesis is dependent on the GPCR. The mouse and human genomic clones may contain the distal and proximal promoters of the GPCRs that will allow the analysis of the transcriptional regulation of hematopoietic-specific genes. Both the mouse and human genomic clones can also be used for cytogenetic mapping to examine whether the GPCRs are linked to any known genetic diseases.

Rabbit antisera was prepared which was reactive with either the N-terminal portion or the C-terminal portion of the receptor as confirmed by ELISA. Two rabbits were injected with a 13 amino-acid peptide corresponding to the cytoplasmic tail of the receptor. Another two rabbits were injected with GST-GPCR-N, a glutathione-S-transferase fusion protein containing the N-terminal extracellular domain of the GPCR. The sera from the second, third, and fourth production bleed of both rabbits exhibited strong immune response to the peptide as seen in the ELISA assay. The antibodies were affinity purified using a peptide affinity column and are valuable for analyzing the expression of this GPCR in T and B cell development.

Monoclonal antibodies to the receptor can also be generated using conventional hybridoma technology known to one or ordinary skill in the art. Briefly, three mice are immunized with 25 .mu.g recombinant receptor prepared as described in Example 9. Mice are inoculated at 3 week intervals with 20 .mu.g GPCR per mouse (1/2 subcutaneously and 1/2 intraperitoneally). Serum collected from each animal after the first inoculation reacts with GPCR as determined by immunoprecipitation. Three days after the final inoculation, mice are sacrificed and the spleens harvested and prepared for cell fusion. Splenocytes are fused with Sp2/0 AG14 myeloma cells (ATCC CRL 1581) with polyethylene glycol (PEG). Following PEG fusion, cell preparations are distributed in 96-well plates at a density of 105 cells per well and selected in hypoxanthine/aminopterin/thymidine (HAT) medium containing 10% fetal calf serum and 100 U/ml interleukin-6. The medium is replaced with fresh HAT medium 10 days after plating. To identify hybridomas producing MAbs which recognize GPCR, hybridoma supernatants are tested for the ability to immunoprecipitate purified recombinant GPCR or to detect GPCR by immunoblotting.

A glutathione-S-transferase (GST) fusion protein of the N-terminal extracellular domain of the GPCR was constructed. The mouse and human GPCRs were cloned into various eukaryotic expression vectors which will allow the overexpression of recombinant mouse and human GPCRs in transfected cells in vitro and in vivo by methods well known to one of ordinary skill in the art. Preferably, the constructs containing the GPCR is transfected into eukaryotic cells; more preferably into mammalian cells. Alternatively, the construct may be used to transform bacterial cells.

Since the GPCR is upregulated by BCR-ABL and can suppress the outgrowth of lymphocytes and fibroblasts (Tables 4A-B), antibodies or drugs can be screened which can activate the action of the GPCR to delay the progression of leukemia. In vitro screening assays can be used to find drugs or natural ligands which bind to and activate the GPCR to delay the progression of leukemia. These drugs or antibodies which inhibit the growth of lymphocytes may also be useful for treatment of diseases such as lymphoma or autoimmune diseases.

Conversely, monoclonal antibodies can be generated against particular regions of GPCRs which block the GPCRs and stimulate the growth of normal lymphocytes in vivo. In addition, in vitro screening assays can be used to find drugs or natural ligands which bind to and either activate or inactivate the GPCR. These antibodies, drugs or natural ligands can stimulate the growth of lymphocytes, which may in turn cure or alleviate the symptoms of patients who have either inherited immunodeficiency diseases or Acquired immune deficiency syndrome (AIDS). For example, patients with severe combined immune deficiency (SCID), DiGeorge syndrome, or Bare lymphocyte syndrome lack T cells, and patients with X-linked agammaglobulinelmia lack B cells. The antibodies, drugs, natural ligands can be delivered into these patients to inhibit the GPCR to stimulate the growth of the T and B cells in their immune system.

In a preferred embodiment, the cDNA encoding the GPCR is placed in a eukaryotic expression vector for taansfection into or infection of a mammalian cell line. Many such cell lines are known in the art, including NIH 3T3, Rat-1, 293T, COS-1, COS-7 and Chinese hamster ovary (CHO) cells, most of which are available from the American type Culture Collection (ATCC), Rockville, Md. Many such expression vectors are known and are commercially available. Preferred expression vectors include retroviral vectors, adenoviral vectors and SV40-based vectors. The vector may contain a selectable marker, such as antibiotic resistance, to select for cells which are expressing the receptor. Alternatively, the expression of the GPCR can be under the control of a regulatory promoter. Stable transfectants are used to screen large libraries of synthetic or natural compounds to identify compounds which bind to the GPCP. Compounds which bind to the GPCR are then tested in the assays to determine whether they are agonists or antagonists of BCR-ABL-mediated GPCR activation.

In one embodiment of the invention, a compound to be tested is radioactively, calorimetrically or fluorimetrically labeled using methods well known in the art and incubated with the receptor. After incubation, it is determined whether the test compound is bound to the receptor. If so, the compound is a potential agonist or antagonist. Functional assays are performed to determine whether the receptor activity is activated or inhibited. These assays include fibroblast and bone marrow transformation assays, cell cycle analysis and in vivo tumor formation assay. Responses can also be measured in cells expressing the receptor using signal transduction systems including, but not limited to, protein phosphorylation, adenylate cyclase activity, phosphoinositide hydrolysis, guanylate cyclase activity, ion fluxes (i.e. calcium) and pH changes. These types of responses can either be present in the host cell or introduced into the host cell along with the receptor.

Because the GPCRs are induced by protein tyrosine kinase oncogenes, they can be used as a diagnostic marker for many types of cancer including leukemia. The DNA sequence can also be used as a probe to search for additional closely-related family members which may play similar roles in oncogenesis.

GPCRs are not expressed in normal bone marrow cells, but are expressed in spleen. Thus, It is possible that GPCRs regulate blood cell development. Regulation of the activity of the GPCR (by antibodies, inhibitory or stimulatory drugs, or natural ligands) may be clinically useful in restoring the normal number and function of the blood cell population with suppressed hematopoiesis, such as that which occurs after treatment to obtain immune depression for organ transplants or after cytotoxic cancer therapy.

The expression of the GPCR in heart suggests that this gene may play a physiological role in heart. It has been shown that there are a variety of autoantibodies, including antireceptor autoantibodies, in patients with cardiomyopathy (Fu, Mol. Cell. Biochem. 163:343-7 (1996). Patients with cardiomyopathy may have autoantibodies against the GPCR which contribute to the pathogenesis of cardiomyopathy. Therefore, regulation of GPCR function by neutralizing antibodies, drugs, or natural ligands may alleviate the symptoms of patients with cardiomyopathy. The GPCRs may also be involved in cardiovascular, hypertension-related, cardiac function defects. Regulation of GPCR function by neutralizing antibodies, drugs, or natural ligands may alleviate the symptoms in patients with such defects.

Since we have isolated both murine and human GPCRs, the cDNAs can be used to isolate the homologue of the GPCRs in other species. Identification of the homologues in other species may lead to a cure for the diseases mentioned above in animals, and will therefore have broad applications in veterinary medicine. The amino acid sequence information of the highly conserved regions of the murine and human GPCRs can be used to develop antibodies or drugs that can be used to treat diseases in both human and animals.

Claim 1 of 6 Claims

What is claimed is:

1. An isolated antibody that specifically binds to the G protein-coupled receptor polypeptide consisting of SEQ ID NO: 2.
 


____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]