WO2000077047A1

WO2000077047A1 - Dog gonadotropin releasing hormone receptor

Info

Publication number: WO2000077047A1
Application number: PCT/US2000/015496
Authority: WO
Inventors: Jisong Cui; Jane-Ling Lo; George R. Mount
Original assignee: Merck & Co., Inc.
Priority date: 1999-06-10
Filing date: 2000-06-06
Publication date: 2000-12-21

Abstract

The dog GnRH receptor has been isolated, cloned and sequenced. The dog GnRH receptor may be used to screen and identify compounds which bind to the GnRH receptor. Such identified compounds may be used in the treatment of sex hormone related conditions such as endometriosis, uterine fibroids, polycystic ovarian disease, hirsutism, precocious puberty, gonadal steroid-dependent neoplasias such as cancers of the prostate, breast and ovary, gonadotrophe pituitary adenomas, sleep apnea, irritable bowel syndrome, premenstrual syndrome and benign prostatic hypertrophy. The receptor proteins and polypeptides, nucleic acids, cells and assays of this invention are useful in drug screening and development, diagnosis and therapeutic applications.

Description

TITLE OF THE INVENTION

DOG GONADOTROPIN RELEASING HORMONE RECEPTOR

FIELD OF THE INVENTION This invention relates to the cloning and isolation of the dog gonadotropin-releasing hormone (GnRH) receptor, and also to mutant or polymorphic forms of the receptor and recombinant nucleic acids encoding the same. The invention also relates to genetically engineered host cells which express the receptor, antibodies against the receptor and polypeptides thereof. The invention also relates to uses of the receptor, recombinant nucleic acids and recombinant host cells in drug screening and development, diagnosis and therapeutic applications.

BACKGROUND OF THE INVENTION Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the control of reproduction. It is a neuronal decapeptide hormone released from hypothalamus in a pulsatile manner. GnRH interacts with its receptor on the gonadotropes in the anterior pituitary and which, in turn, activates phospholipase C (PLC) via a pertussis toxin-insensitive G protein, Gq/Gll. Two second messengers, inositol trisphosphate (IP3) and diacylglycerol (DG), are formed from the hydrolysis of phosphoinositide bisphosphate by PLC. IP3 and DG then act either separately or in concert, via increase of intracellular Ca²⁺ and activation of protein kinase C, to regulate the synthesis and release of both luteinizing hormone (LH) and follicle stimulating hormone (FSH). LH released from the pituitary gland is primarily responsible for the regulation of gonadal steroid production in both sexes, whereas FSH regulates spermatogenesis in males and follicle development in females.

The GnRH receptor (GnRH-R) is mainly expressed in the pituitary gland. It was also detected in extrapituitary tissues such as brain, breast, gonads, and ovarian tumors. The GnRH receptor has been cloned and sequenced from several mammalian species including human, ovine, bovine, pig, rat, and mouse. The cloning and expression of the murine and human receptors has recently been described in U.S. Patent No 5,750,366 The GnRH receptor from bovine, cow, sheep, and human contains 328 ammo acids, while the rodent receptor has 327 ammo acids, due to a deletion of a residue in the second extracellular domain Analysis of the primary sequence identifies the GnRH receptor as a member of the G protem-coupled receptor (GPCR) family with seven transmembrane (TM) domains However, the mammalian GnRH receptors have several unique structural features compared with other GPCRs These include (1) the lack of the entire intracellular C-terminal tail, (2) the replacement of Tyr by Ser m the conserved G protein signature DRY motif of the proximal second intracellular domain, and (3) the reciprocal exchange of two ammo acids, Asp in TM II and Asn in TM VII, that are highly conserved in most other GPCRs In addition to pituitary gland, the expression of GnRH receptor message has also been demonstrated in extrapituitary tissues such as brain, breast, gonads, and ovarian tumors The receptor sequences obtained from extrapituitary sources were identical to the corresponding pituitary GnRH receptor cDNAs.

Synthetic peptidyl GnRH analogues have been widely used in treating patients with prostate and breast cancers, endometnosis, uterine fibroids, precocious puberty, and other endocrinological disorders. Continuous infusion of high dose GnRH agonists internalizes the GnRH receptor and subsequently desensitizes the receptor, therefore effectively reducing the levels of LH and FSH. However, GnRH agonists can cause flare of the disease at the initial use due to their stimulatory effects. Cetrorelix and other peptide antagonists which are under development have been shown to reduce the levels of LH and FSH without the flare effect Non-peptidyl GnRH antagonists have been disclosed m U.S. Patent Nos 5,756,507, 5,780,437 and 5,849,764.

Although dogs have been used to evaluate the efficacy of GnRH agonists and antagonists in vivo, the dog GnRH receptor has not been cloned and expressed in any cell lines. In this invention, we have cloned and sequenced the dog GnRH receptor and functionally expressed the receptor in a mammalian cell system. These tools facilitate the development of better GnRH agonists or antagonists for treating certain GnRH derived disorders SUMMARY OF THE INVENTION

This invention relates to the cloning and sequencing of the dog GnRH receptor. The DNA sequences disclosed herein may be engineered into expression systems designed for the production of the receptor and/or cell lines which express the receptor. Such cell lines may be used for screening and identifying compounds that function as GnRH agonists and antagonists.

Other aspects of this invention are nucleic acids which encode the receptor or a functional equivalent. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. For most cloning purposes, cDNA is a preferred nucleic acid, but this invention specifically includes other forms of DNA as well as RNAs which encode the receptor or a functional equivalent. A further aspect of this invention relates to vectors which comprise nucleic acids encoding the dog GnRH receptor or a functional equivalent. These vectors may be comprised of DNA or RNA; however, for most cloning purposes, DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes and other forms of episomal or integrated DNA that can encode the receptor. It is well within the skill of the ordinary artisan to determine an appropriate vector for a particular gene transfer or other use.

Other aspects of this invention are host cells which are transformed with a gene which encodes the dog GnRH receptor or a functional equivalent. The host cell may or may not naturally express the receptor on the cell membrane. Preferably, once transformed, the host cells are able to express the receptor or a functional equivalent on the cell membrane. Depending on the host cell, it may be desirable to adapt the DNA so that particular codons are used in order to optimize expression. Such adaptations are known in the art, and these nucleic acids are also included within the scope of this invention. Generally, mammalian cell lines, such as COS, HEK-293, CHO, HeLa, NS/O, CV-1, GC, GH3 or VERO cells are preferred host cells, but other cells and cell lines such as Xenopus oocytes or insect cells, may also be used.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 shows the primer sequences used in the invention, as set forth in SEQ ID Nos. 1-8.

JC 1-1; 5'-ACTCGTCGACAAYCAYTGYAGYGCNATHAA-3' JC 3; 5'-ACTCGAATTCTACCAYTGNACNGTDATRTTCCA CATNCC-3'

JC 5; 5'-ACTCGTCGACAARATGACNGTNGCNTTYGC-3' JC 8; 5'-CTACAAAGAAAARTANCCRTADATNAGNGGRTC-3'

Dog l 5'-ACTGTCACTCGGATCTTC-3' Dog 3 5'-TGTCAGATCCAGTAAATCAC-3' Dog 6 5'-ACTCGAATTCGCCACCATGGCAAGCGCCTCTCC-3' Dog 7 5'-ACTCTCTAGATTACAGAGAGAAATATCC-3' (Y=C/T; N=A/C/T/G; H=A/T/C; D=A/T/G; R=A/G)

FIGURE 2 is the DNA of the dog GnRH receptor that encodes the entire open reading frame, from Met Initiation codon to the Stop codon as set forth in SEQ ID no. 9.

FIGURE 3 is the amino acid sequence of the dog GnRH receptor encoded by the DNA in FIGURE 2, as set forth in SEQ ID no. 10.

FIGURE 4 is a graph demonstrating the expression of the dog GnRH receptor in transfected CHO-Kl cells using the membrane binding assay.

FIGURE 5 is a graph demonstrating the G-protein coupling of the dog GnRH receptor in transfected CHO-Kl cells using the PI turnover assay.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the cloning of the dog gonadotropin- releasing hormone (GnRH) receptor, and also to mutant or polymorphic forms of the receptor and recombinant nucleic acids encoding the same. The invention also relates to genetically engineered host cells which express the receptor as well as antibodies against the receptor and polypeptides thereof. The invention also relates to uses of the receptor, recombinant nucleic acids and recombinant host cells in drug screening and development, diagnosis and therapeutic applications.

Each document mentioned in this specification is hereby incorporated herein by reference in its entirety.

As used herein a "compound" or a "molecule" is an organic or inorganic assembly of atoms of any size, and can include macromolecules, e.g., peptides, polypeptides, whole proteins, and polynucleotides. The terms are used interchangeable herein.

As used herein, a "candidate" is a molecule or compound that may be an modulator, agonist or antagonist of the receptor.

As used herein an "agonist" is a compound or molecule that interacts with and activates a polypeptide of the receptor.

As used herein an "antagonist" is a compound or molecule that interacts with and inhibits or prevents a polypeptide of the receptor from becoming activated.

As used herein a "modulator" is a compound or molecule that interacts with an aspect of cellular biochemistry to effect an increase or decrease in the amount of a polypeptide of the receptor present at the surface of a cell, or in the surrounding serum or media. The change in amount of the receptor polypeptide can be mediated by the effect of a modulator on the expression of the receptor, e.g., the transcription, translation, post-translational processing, translocation or folding of the receptor, or by affecting a component(s) of cellular biochemistry that directly or indirectly participates in the expression of the receptor. Alternatively, a modulator can act by accelerating or decelerating the turnover of the receptor either by direct interaction with the receptor or by interacting with another component(s) of cellular biochemistry which directly or indirectly effects the change.

As used herein a "polynucleotide" is a nucleic acid of more than one nucleotide. A polynucleotide can be made up of multiple poly- nucleotide units that are referred to by description of the unit. For example, a polynucleotide can comprise within its bounds a polynucleo- tide(s) having a coding sequence(s), a polynucleotide (s) that is a regulatory region(s) and/or other polynucleotide units commonly used in the art. An "expression vector" is a polynucleotide having regulatory regions operably linked to a coding region such that, when in a host cell, the vector can direct the expression of the coding sequence. The use of expression vectors is well known in the art. Expression vectors can be used in a variety of host cells and, therefore, the regulatory regions are preferably chosen as appropriate for the particular host cell.

A "regulatory region" is a polynucleotide that can promote or enhance the initiation or termination of transcription or translation of a coding sequence. A regulatory region includes a sequence that is recognized by the RNA polymerase, ribosome, or associated transcription or translation initiation or termination factors of a host cell. Regulatory regions that direct the initiation of transcription or translation can direct constitutive or inducible expression of a coding sequence.

The isolated nucleic acid molecule of the present invention can include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which can be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention can also include a ribonucleic acid molecule (RNA). The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.

Polynucleotides of this invention contain full length or partial length sequences of the receptor gene. Polynucleotides of this invention can be single or double stranded. If single stranded, the polynucleotides can be a coding, "sense," strand or a complementary, "antisense," strand. Antisense strands can be useful as modulators of the receptor by interacting with RNA encoding the receptor. Antisense strands are preferably less than full length strands having sequences unique or highly specific for RNA encoding the receptor.

The polynucleotides can include deoxyribonucleotides, ribonucleotides or mixtures of both. The polynucleotides can be produced by cells, in cell-free biochemical reactions or through chemical synthesis. Non-natural or modified nucleotides, including inosine, methyl-cytosine, deaza-guanosine, etc., can be present. Natural phosphodiester internucleotide linkages can be appropriate. However, polynucleotides can have non-natural linkages between the nucleotides. Non-natural linkages are well known in the art and include, without limitation, methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites and phosphate ester linkages. Dephospho-linkages are also known, as bridges between nucleotides. Examples of these include siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bridges. "Plastic DNA," having, for example, N-vinyl, methacryloxytethyl, methacrylamide or ethyleneimine internucleotide linkages, can be used. "Peptide Nucleic Acid" (PNA) is also useful and resists degradation by nucleases. These linkages can be mixed in a polynucleotide.

As used herein, "purified" and "isolated" are utilized interchangeably to stand for the proposition that the polynucleotides, proteins and polypeptides, or respective fragments thereof in question has been removed from its in vivo environment so that it can be manipulated by the skilled artisan, such as but not limited to sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragment in pure quantities so as to afford the opportunity to generate polyclonal antibodies, monoclonal antibodies, amino acid sequencing, and peptide digestion. Therefore, the nucleic acids claimed herein can be present in whole cells or in cell lysates or in a partially purified or substantially purified form. A polynucleotide is considered purified when it is purified away from environmental contaminants. Thus, a polynucleotide isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.

It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA sequences encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown below:

A=Ala=Alanine: codons GCA, GCC, GCG, GCU

C=Cys=Cysteιne: codons UGC, UGU

D=Asp=Aspartic acid: codons GAC, GAU

E=Glu=Glutamic acid: codons GAA, GAG

F=Phe=Phenylalanine: codons UUC, UUU G=Gly=Glycine: codons GGA, GGC, GGG, GGU

H=His=Histidine: codons CAC, CAU

I=Ile=Isoleucine: codons AUA, AUC, AUU

K=Lys=Lysine: codons AAA, AAG

L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU M=Met=Methionine: codon AUG

N=Asp^:=Asparagine: codons AAC, AAU

P=Pro=Proline: codons CCA, CCC, CCG, CCU

Q=Gln=Glutamine: codons CAA, CAG

R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU

T=Thr=Threonine: codons ACA, ACC, ACG, ACU

V=Val=Valine: codons GUA, GUC, GUG, GUU

W=Trp=Tryptophan: codon UGG

Y=Tyr=Tyrosine: codons UAC, UAU

Therefore, the present invention discloses codon redundancy which can result in differing DNA molecules expressing an identical protein. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide. It is known that DNA sequences coding for a peptide can be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide. Methods of altering the DNA sequences include but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.

As used herein, a "biologically active equivalent" or "functional derivative" of a wild-type dog GnRH receptor possesses a biological activity that is substantially similar to the biological activity of the wild type dog receptor. The term "functional derivative" is intended to include the "fragments," "mutants," "variants," "degenerate variants," "analogs" and "homologues" or to "chemical derivatives" of the wild type dog receptor. The term "fragment" is meant to refer to any polypeptide subset of wild-type dog receptor. The term "mutant" is meant to refer to a molecule that may be substantially similar to the wild-type form but possesses distinguishing biological characteristics. Such altered characteristics include but are in no way limited to altered substrate binding, altered substrate affinity and altered sensitivity to chemical compounds affecting biological activity of the dog receptor or dog receptor functional derivative. The term "variant" is meant to refer to a molecule substantially similar in structure and function to either the entire wild- type protein or to a fragment thereof. A molecule is "substantially similar" to a wild-type dog receptor if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical. The term "analog" refers to a molecule substantially similar in function to either the full-length dog receptor or to a biologically active fragment thereof.

A protein or fragment thereof is considered purified or isolated when it is obtained at a concentration at least about five-fold to ten-fold higher than that found in nature A protein or fragment thereof is considered substantially pure if it is obtained at a concentration of at least about 100-fold higher than that found in nature A protein or fragment thereof is considered essentially pure if it is obtained at a concentration of at least about 1000-fold higher than that found m nature Probes can be labeled by any number of ways known in the art including isotopes, enzymes, substrates, chemilummescent, electrochemilummescent, biotm and fret pairs among many others A probe so labeled can generate a detectable signal directly (e.g., isotopes), or upon hybridization (fret pairs), or indirectly after a chemical (e g , luminescence) or biochemical reaction (e g., enzyme-substrate) or after binding a strepavidin linked moiety that can generate a detectable signal direclty or indirectly. The labeling of probes and the generation of detectable signals are well known techniques in the art and include Polymerase Chain Reaction and Reverse Transcπptase Polymerase Cham Reaction (See e.g., PCR Primer, edited by C W Dieffenbach and G S Dveksler, (1995) Cold Spring Harbor Laboratory Press.), Strand Displacement Amplification, Self-Sustained Sequence Reaction, and any other amplification known to one of skill in the art that uses primers

Expression of the receptor

The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification. Therefore, the present invention also relates to methods of expressing the dog GnRH receptor and biological equivalents disclosed herein, assays employing these recombinantly expressed gene products, cells expressing these gene products, and modulators, agonistic and/or antagonistic compounds identified through the use of assays utilizing these recombinant forms, including, but not limited to, one or more compounds or molecules that act through direct contact with the receptor, particularly with the ligand binding domain, or through direct or indirect contact with a ligand which either interacts with the receptor or with the transcription or translation of GnRH, thereby modulating GnRH expression.

A variety of expression vectors can be used to express the dog GnRH receptor in host cells. Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.

Commercially available mammalian expression vectors which can be suitable for the dog GnRH receptor expression, include but are not limited to, pcDNA3.1 (Invitrogen), pIRESl/ieo (Clontech), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593) pBPV- 1(8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).

A variety of bacterial expression vectors can be used to express the dog GnRH receptor in bacterial cells. Commercially available bacterial expression vectors which are suitable for recombinant dog GnRH receptor expression include, but are not limited to pQE (Qiagen), pETlla (Novagen), lambda gtll (Invitrogen), and pKK223-3 (Pharmacia).

A variety of fungal cell expression vectors can be used to express the dog GnRH receptor in fungal cells. Commercially available fungal cell expression vectors which are suitable for the dog GnRH receptor expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).

A variety of insect cell expression vectors can be used to express recombinant receptor in insect cells. Commercially available insect cell expression vectors which are suitable for the dog GnRH receptor include but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).

An expression vector containing DNA encoding the dog GnRH receptor can be used for expression of the dog GnRH receptor in a recombinant host cell. Recombinant host cells can be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines. Cell lines derived from mammalian species which can be suitable and which are commercially available, include but are not limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209).

The expression vector can be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation. The expression vector-containing cells are individually analyzed to determine whether they produce the dog GnRH receptor. Identification of the dog GnRH receptor expressing cells can be done by several means, including but not limited to immunological reactivity with anti-dog GnRH antibodies, labeled ligand binding and the presence of host cell-associated GnRH activity

The cloned dog GnRH receptor cDNA obtained through the methods described herein can be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3 1, pIRESlzieo, pQE, pBlueBacHιs2 and pLITMUS28) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant human HG38 Techniques for such manipulations can be found described in Sambrook, et al , supra , and are well known and easily available to the one of ordinary skill in the art

EXAMPLE 1

mRNA Isolation and cDNA Synthesis

Total RNA from dog pituitaries (snap-frozen in liquid nitrogen within 1-2 minutes of animal sacrifice, Rockland, Gilbertsville, PA) was prepared using the TRIzol reagents (Promega, Madison, WI) following the manufacturer's instruction Typically, 0 5 mg of total RNA was isolated from 350 mg wet weight of pituitary tissue Poly (A) RNA was isolated from total RNA by column chromatography on oligo (dT) cellulose (Promega, Madison, WI) The yield of poly (A) mRNA from total RNA was usually 0.5%.

First-strand cDNA was synthesized from poly (A)⁺ mRNA using Superscript II reverse transcπptase (GIBCO-BRL, Grand Island, NY) as per the manufacture's instruction One-tenth of the volume was used for each RT-PCR reaction.

EXAMPLE 2

Cloning of the Dog GnRH Receptor cDNA The steps for cloning the dog GnRH receptor cDNA included: (1) isolation of the genomic clones of the dog GnRH receptor; (2) PCR amplifying partial coding sequences from the genomic clones by degenerate primers; and (3) cloning of the entire open reading frame (ORF) of the dog receptor by RT-PCR from the dog pituitary mRNA.

1. Isolation of the dog GnRH receptor genomic clones

The genomic clones that contained the dog GnRH receptor were pulled out from a dog genomic library (Stratagene, La Jolla, CA) under low stringency hybridization conditions. Approximately 10⁶ phage plaques of a dog genomic library in Lambda DASH were plated on the E. coli strain XLl-Blue MRA (Stratagene, La Jolla, CA). The plaques were transferred to Nitrocellulose membranes (Schleicher & Schuell, Keene, NH), denatured, neutralized, and screened with a 1 kb cDNA of the human GnRH receptor (Kakar et al., 1992, Biochem. Biophys. Res. Commun. 189, 289-295). The membranes were incubated at 42°C in prehybridization solution (40% formamide, 1 x Denhardts, 6 x SSC, 0.5% SDS, 100 μg/ml salmon sperm DNA) for 3 hours followed by overnight incubation in hybridization solution (40% formamide, 1 x Denhardts, 6 x SSC, 100 μg/ml salmon sperm DNA) with 1 x 10⁶ cpm/ml of [³²P]-labeled probe. The probe was labeled with [³²P]-dCTP using a random priming kit (Amersham, Arlington Heights, IL). After hybridization, the membranes were first washed three times with 2 x SSC, 0.1% SDS at room temperature, and finally washed once with 0.5 x SSC, 0.1% SDS at 50°C for 30 minutes. A single positive clone was isolated following three rounds of plaque purification.

Phage containing the dog GnRH receptor was eluted from plate plaques with 1 x lambda buffer (0.1 M NaCl, 10 mM MgSO₄7H₂O, 35 mM Tris-HCl, PH7.5) following overnight growth of approximately 10¹⁰ pfu/150 mm dish. After a 10 minute centrifugation at 10,000 x g to remove debris, the phage solution was treated with 1 mg/ml RNAse A and DNAse I for 30 minutes at 24°C, followed by precipitation with 20% PEG (8000)/2 M NaCl for two hours on ice, and collection by centrifugation at 10,000 x g for 20 minutes. Phage DNA was isolated by incubation in 0.1% SDS, 30 mM EDTA, 50 mg/ml proteinase K for one hour at 68°C, with subsequent phenol (three times) and chloroform (twice) extraction before isopropanol precipitation overnight. The inserts in lambda DASH were individually sub-cloned into the plasmid vector pBlueScπpt (GIBCO- BRL. Grand Island, NY). 2 μg of phage DNA was heated to 65°C for 10 minutes, then digested with 10 units of Sail at 37°C for 4 hours. A fragment of 10 kb (containing exon 1) or 12 kb (containing exon 3) was subcloned into vector pBlueScript.

2. Cloning of Partial cDNAs of the Dog GnRH Receptor

The ORF of human GnRH receptor spins three exons (1-3) on the genome. The exon 1 contains the translational initiator and the exon 3 contains the stop codon (Fan et al., 1994, Molecular and Cellular Endocrinology. 103, R1-R6). Since the gene organization across species is well conserved, it was assumed that the dog GnRH receptor is also contained in three exons.

Degenerate primers were designed based on the sequences of cloned GnRH receptors from various mammalian species. The PCR was performed on the above phage clones using PCR kit (GIBCO-BRL, Grand Island, NY) under the condition of 94°C for 80 seconds, 50°C for 1 minute, and 72°C for 2 minutes (30 cycles) with the final extension at 72°C for 10 minutes. PCR products of partial exon 1 (by primers of JCl-1 + JC3, Figure 1) and exon 3 (by primers JC5 + JC8, Figure 1) were obtained. The PCR products were extracted by equal volume of phenol/chloroform, followed by ethanol precipitation. The purified DNA fragments were individually ligated into the plasmid vector pCR2.1 (Invitrogen Corporation, San Diego, CA) by T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN), and transformed into the DH5α cells (GIBCO-BRL, Grand Island, NY). Plasmid DNA was prepared by Qiagen kit (Qiagen Inc., Valencia, CA). The cloned cDNAs were sequenced (ACGT, Inc., Northbrook, Illinois) at both strands. Based on these sequences, primers Dog 1 and Dog 3 (Figure 1) were designed and used to determine the 5' and 3' sequences of the dog GnRH receptor from these genomic clones.

3. Cloning and sequencing of the full-length coding cDNA of the dog GnRH receptor The full-length coding cDNA of the dog GnRH receptor was cloned from dog pituitary mRNA by RT-PCR using primers Dog 6 (5') + Dog 7 (3') (Figure 1). The PCR was performed using PCR kit (GIBCO- BRL, Grand Island, NY) under the condition of 94°C for 80 seconds, 60°C for 1 minute; and 72°C for 2 minutes (30 cycles) with the final extension at 72°C for 10 minutes. The PCR product had expected size of ~ 1 kb. This product was extracted by equal volume of phenol/chloroform, followed by ethanol precipitation. The purified DNA fragment was directly ligated into the plasmid vector pCR2.1 (Invitrogen Corporation, San Diego, CA) by T4 DNA ligase (Boehringer Mannheim, Indianapolis, IN), and transformed into the DH5α cells (GIBCO-BRL, Grand Island, NY).

Plasmid DNA was prepared by Qiagen kit (Qiagen Inc., Valencia, CA). The DNA of the dog GnRH receptor was sequenced (ACGT, Inc., Northbrook, Illinois) at both strands. Four clones that were derived from independent PCRs had identical sequences (Figure 2).

EXAMPLE 3

The Dog GnRH Receptor

Data searches (Genbank 88, EMBL 42, Swiss-Prot 31, PIR

40, dEST, Prosite), sequence alignments and analysis of the dog GnRH receptor nucleotide and protein sequences were carried out using the GCG Sequence Analysis Software Package (Madison, WI; pileup, peptide structure and motif programs), FASTA and BLAST search programs, and PC/Gene software suite from Intelligenetics (San Francisco, CA; protein analysis programs).

The dog receptor contains 327 amino acids (Figure 3), one residue shorter than that of the human receptor. Surprisingly, the dog receptor lacks an asparagine equivalent to the third residue of the human receptor. The asparagine residue at this position is well conserved in all other known GnRH receptors of mammalian species.

The dog receptor DNA has 91% homology with the human GnRH receptor, and its protein has 92.6% identity and 96.6% similarity with the human receptor. The dog GnRH receptor has the characteristic of the G-protein coupled receptors with seven putative tramsmembrance domains. The sites for glycosylation and for disulfide bond formation are well conserved compared with other mammalian GnRH receptors. Similar to the human receptor, the dog GnRH receptor also lacks a C-terminal cytoplamic tail.

EXAMPLE 4

Generation of the CHO-Kl Cells That Stably Express the Dog GnRH Receptors

The coding cDNA of the dog GnRH receptor was subcloned into a polycistronic expression vector pIRESl. π,eo (Clontech Laboratories, Palo Alto, CA) to generate cells stably expressing the receptor. The plasmid DNA of pIRESl./ieo-dog-GnRHR was transfected into CHO-Kl cells by lipofectin (GIBCO-BRL, Grand Island, NY) following the manufacturer's instruction. Transfection was carried out overnight and the cells were split 48 hours later. G418 at 1 mg/ml was added 24 hours after splitting, and its concentration was increased to 2 mg/ml in 2-3 days. After 10 days selection by G418, colonies were picked and transferred to 24-well plates. The clones that expressed the dog GnRH receptor were screened by a whole cell binding assay using a radiolabeled peptide agonist of GnRH, 5-(¹²⁵Iodo-Tyr)-Buserelin (Woods Assay, Portland, Oregon). Seven of the 71 neo^ clones screened had a specific binding activity of 5-fold above the non-specific binding. The best one, 1B2-1F9 had a specific binding activity of more than 10-fold above the background. After single cell isolation, the membrane from this clone was prepared, and the membrane binding assay was performed using ¹²⁵I-buserelin. Figure 4 shows that the ligand binding activity was dose-dependent. The GnRH-stimulated PI turnover was also performed. 1B2-1F9 responded to GnRH stimulation also in a dose-dependent manner with an EC50 value of 0.3-0.4 nM (Figure 5). This result demonstrated that these cells were functionally coupled with G-proteins.

EXAMPLE 5

Bioassays

Membrane binding assay

The ligand binding assay was conducted with the dog GnRH receptor obtained from 1B2-1F9. Crude membrane suspensions were prepared from 1B2-1F9 cells and stored as aliquots at -80°C. The radioactive peptide ligand [5-(^12δIodo-Tyr)-Buserelin] was obtained from Woods Assay (Portland, Oregon) and had a radioactive specific activity of 1000 Ci/mmol. The membranes and radioligand were diluted in assay buffer which consists of 50 mM Tris-HCl (PH 7.5), 2 mM MgCl₂ and 0.1% bovine serum albumin. The ligand binding assay was performed at 22°C for 1 hour in borosilicate glass tubes in a final volume of 500 ml. Each tube contained an appropriate concentration of the radioligand (from 0.025 to 2.5 nM) and 15-20 mg of 1B2-1F9 membrane protein. The incubation was terminated by vacuum filtration onto GF/C Whatman filter paper (pretreated with 0.1% BSA in PBS) by a Brandel 48-well Harvester and then washed with 2 ml of cold phosphate saline (PH 7.5). The filter strips were counted in a Gamma Counter.

Phosphoinositide (PI) turnover assay

1B2-1F9 cells are functionally coupled to phospholipase C. To perform the PI turnover assay, 1B2-1F9 cells were seeded into 24-well plates at a concentration of 80,000 cells/ml/well in inositol-free F12 medium (Gibco Life Technology, Grand Island, NY) containing 10% dialyzed fetal bovine serum (Gibco Life Technology, Grand Island, NY), 1% Pen/Strep, 2 mM glutamine, 2 mg/ml G418 and 1 μCi (³H) inositol (NEN Life Sciences, Boston, MA). Forty-eight hours after seeding, cells were washed with 3 x 1 ml of PBS containing 10 mM LiCl and treated with various concentrations of GnRH (Biochem, Torrance, CA ). After incubation at 37°C for 1 hour, the medium was removed and the cells were lysed with 1 ml of 0.1 M formic acid. The plates were freeze-thawed once and the cell extract was applied onto a Dowex AG1-X8 column (Bio- Rad Labs, Hercules, CA). The column was washed with 2 x 1 ml H₂O to remove free (³H)inositol, and (³H)inositol phosphates were eluted with 3 x 1 ml 2 M ammonium formate in 1 M formic acid. The eluate was then counted in a scintillation counter.

Claims

WHAT IS CLAIMED IS:

1. The dog GnRH receptor, free from receptor- associated proteins.

2. Isolated dog GnRH receptor.

3. A purified DNA molecule encoding the dog GnRH receptor which comprises the nucleotide sequence:

atggcaagcg cctctcctga acagaatcaa aatcactgct cagctgtaaa caacagcaac atgctgatgc agggcaacct ccccaccctg accttatctg ggaagatccg agtgacagtt actttcttcc tttttcttct ctctacaatt tttaatgctt ctttcttgtt gaaacttcag aagtggactc aaaagaaaga gaaagggaag aagctctcga gaatgaaagt gcttttaaaa catctgacct tggccaacct gttggagacc ctgattgtca tgccactgga tggaatgtgg aacattacag tccaatggta tgctggagag ttcctctgca aagtcctcag ctatctgaag cttttctcca tgtatgcccc agccttcatg atggtagtga tcagcctgga ccgctccctg gccatcacga ggcctctagc tatgaaaaac aatggcaagc tcggacagtc catgattggc ctggcctggc tcctcagtgg tatctttgct ggaccacagt tgtacatctt caggatgatc cacttagcag atagctctgg acagacagag ggtttccccc aatgtgtaac acactgcagt tttccacaat ggtggcatca agccttttat aacttcttca ccttcagctg cctcttcatc atccctcttt tcatcacgtt aatctgcaat gcaaaaatca tatttactct gacacgagtc cttcatcagg atccccatga attacaactg aatcagtcca agaataatat accaagagct cggctgagga ccttgaaaat gacagttgca tttgccactt cgtttactgt gtgctggact ccctactatg ttctaggaat ttggtactgg tttgatcctg aaatgttaaa cagggtgtca gatccagtaa atcacttctt ctttcttttt gctcttttaa atccatgctt tgatccactt atatatggat atttctctct gtaa, disclosed herein as

SEQ ID No.9.

4. A purified DNA molecule encoding the dog

GnRH receptor wherein said DNA molecule encodes a protein comprising the amino acid sequence:

MASASPEQNQ NHCSAVNNSN MLMQGN PTL T SGKIRVTV TFF FLLSTI

FNASFLLKLQ K TQKKEKGK K SRMKVLLK HLT AN ET LIVMP DGMW

NITVQ YAGE FLCKVLSYLK LFSMYAPAFM MWISLDRSL AITRPLAMK

NGKLGQSMIG LA LLSGIFA GPQLYIFRMI H ADSSGQTE GFPQCVTHCS FPQ HQAFY NFFTFSCLFI IP FITLICN AKIIFTLTRV HQDPHELQL NQSKNNIPRA RLRT KMTVA FATSFTVCWT PYYVLGIWYW FDPEMLNRVS DPVNHFFFLF ALLNPCFDPL IYGYFSL* , disclosed herein as SEQ ID No.

10.

5. An expression vector for the expression of the dog GnRH receptor in a recombinant host cell wherein said expression vector comprises a DNA molecule which encodes the amino acid sequence of Claim 4.

6. The expression vector according to Claim 5 which is selected from the group consisting of plasmids, modified viruses, yeast artificial chromosomes, bacteriophages, cosmids and transposable elements.

7 A host cell which expresses a recombinant dog

GnRH receptor wherein said host cell contains the expression vector of Claim 5.

8. A host cell which expresses a recombinant dog

GnRH receptor wherein said host cell contains the expression vector of Claim 6.

9. A method for determining whether a substance is a potential antagonist of the dog GnRH receptor comprising:

(a) transfectmg or transforming cells with an expression vector of claim 5 that directs expression of the dog GnRH receptor in the cells, resulting in test cells,

(b) allowing the test cells to grow for a time sufficient to allow the dog GnRH receptor to be expressed,

(c) exposing the cells to a labeled ligand of the dog GnRH receptor in the presence and in the absence of the substance,

(d) measuring the binding of the labeled ligand to the dog GnRH receptor, where if the amount of binding of the labeled ligand is less m the presence of the substance than m the absence of the substance, then the substance is a potential antagonist of the dog GnRH receptor.