JPH05271285A - Protein, dna and its use - Google Patents

Abstract

PURPOSE:To provide a new protein useful for the investigation of human ovarian physiology, diagnosis of ovarian or testicular diseases such as human ovulation disorder and oligospermia, development of anticonceptive agent and therapeutic agent and examination agent for man. CONSTITUTION:A human luteinizing hormone-human chorionic gonadotropin receptor (human LH/hCG receptor) protein. The protein can be produced by culturing a transformant integrated with a DNA containing a cDNA coding the human LH/hCG receptor protein and separating the human LH/hCG receptor protein produced and accumulated in the culture product. The transformant can be prepared e.g. by separating an mRNA from a cell capable of producing human LH/hCG receptor, synthesizing a cDNA from the mRNA, integrating the cDNA into a plasmid and transforming a host with the recombinant plasmid.

Description

Detailed Description of the Invention

[0001]

The present invention relates to human luteinizing hormone-
Human chorionic gonadotropin receptor protein (human LH / h
D containing a cDNA encoding a CG receptor protein)
NA, human LH / hCG receptor protein and method for producing the protein.

[0002]

Luteinizing hormone-human chorionic gonadotropin receptor protein (LH / hCG receptor protein) is
It is present in testicular Leydig cells, ovarian capsule cells, granulosa cells, luteal cells and stromal cells, and plays a central role in reproductive physiology. In males and non-pregnant females, the LH / hCG receptor is affected only by luteinizing hormone, which is produced and secreted in the anterior pituitary. However, in pregnant females, the ovarian LH / hCG receptor is a human chorionic gonadotropin (hC) produced by the placenta.
It will also be affected by G). LH and hCG
Is one of the glycoprotein hormones of which thyroid stimulating hormone (TSH) and follicle stimulating hormone (FSH) are also members. These four hormones have a molecular weight of 28- to 38-kD and are heterodimeric with a specific β subunit associated with receptor binding specificity bound to a common α subunit. It is a glycoprotein. The sugar moieties of these hormones appear to play an important role in signal transduction. Β for both LH and hCG
The subunits are closely related in their structure,
These two hormones bind to the same receptor and trigger the same biological response. Given the similarity of these glycoprotein hormones and the property that these hormones act on their receptors to increase the G-protein mediated adenylyl cyclase activity, the structure of these receptors is dependent on the hormone-inducing activity. It can be seen that they have a common mechanism of action. This increase in adenosine 3 ', 5'-monophosphate (cyclic AMP) inevitably leads to steroid synthesis and secretion. Receptors coupled to G-proteins share the common structural feature of having seven transmembrane domains known to be involved in signal transduction and binding to small ligands. Have been identified as On the other hand, TS
When the H and FSH receptors were compared with the LH / hCG receptors, the receptors for these pituitary glycoprotein hormones were 7
It is also characterized within the G-protein coupled receptor by the presence of a large glycosylation domain that is presumably located extracellularly grafted to a structure containing two transmembrane moieties. The elucidation of the structure of the LH / hCG receptor has been less successful so far in rats and pigs due to its very low abundance and its sensitivity to proteolysis. For the LH / hCG receptors of E. coli, the complementation D of these receptors
NA (cDNA) has been isolated, and the amino acid sequence has been deduced from this DNA [rat: Science]
245 , 494 (1989), Pig: Science 245 , 5
25 (1989)].

[0003]

Thus, the structures of rat and pig LH / hCG receptors have been elucidated, but the structures of human LH / hCG receptors have not been elucidated. When it was considered to be used as a human therapeutic drug or test drug, it was necessary to clarify the structure and properties of human LH / hCG receptor.

[0004]

[Means for Solving the Problems] If the present inventors could collect LH / hCG receptor from humans and produce it by gene recombination technology, it would have a great effect on future research and treatment. As a result of repeated studies, it was confirmed that rat LH / hCG receptor-complementary DN
C that encodes the human LH / hCG receptor for the first time from a human ovarian cDNA library using A as a probe
We have succeeded in cloning the DNA and elucidating the complete nucleotide sequence. Furthermore, from this cDNA, human L
The inventors have succeeded in clarifying the amino acid sequence of the H / hCG receptor and paving the way for mass production of this by gene recombination technology. This receptor is very similar to that of rat and pig, but differences are recognized to the extent that each receptor is recognizable as distinct. The present invention provides (1) human luteinizing hormone-human chorionic gonadotropin receptor protein, (2) human luteinizing hormone-human chorionic gonadotropin receptor protein, and cD
DNA containing NA, (3) human luteinizing hormone-
C encoding the human chorionic gonadotropin receptor protein
A transformant retaining DNA containing DNA, and (4) human luteinizing hormone-human chorionic gonadotropin including culture of the transformant of (3) above, production accumulation of protein in the culture, and collection The present invention relates to a method for producing a receptor protein.

The present inventor cloned two types of human luteinizing hormone-human chorionic gonadotropin receptor protein cDNA, and estimated the primary structure of the complete protein (FIG. 1). The first methionine in this sequence is considered the initiation codon. Following this is an amino acid sequence characteristic of a signal peptide with a cleavage site. Hydropathy analysis
And comparison with rat, porcine LH / hCG receptors (Fig. 2) suggested a possible model for the organization of the protein. The deduced extracellular domain of 335 amino acids precedes a 267 amino acid region representing the seven deduced transmembrane regions (boxed in Figure 2).
There is a 72 amino acid COOH-terminal intracellular domain. This mature protein consists of 674 amino acids (75632 daltons). In addition to this, there are 25 signal peptides (amino acids 1 to 25 in FIGS. 1 and 2), which are cleaved off during receptor synthesis to form 674 amino acids (26 to 26 amino acids) as a receptor maturation protein. 699th). At the level of primary structure, this extracellular domain has approximately 85% identity with rat and porcine LH / hCG receptors and 45% identity with TSH and FSH receptors (Fig. 2). , HLH / hC
GR: human LH / hCG receptor, rLH / hCGR: rat LH / hCG receptor, pLH / hCGR: porcine LH
/ HCG receptor, hTSHR: human TSH receptor [Biochemical. Biophys. Res. Comm.] 16
6 , 394 (1990)], rFSHR: rat FSH receptor [Molecular Endocrinology (Mol. Endo.) 4 , 525]
(1990)]). Six potential glycosylation sites are found in the putative extracellular domain (Figure 1, underlined). Population of cysteine is present between the extracellular domains and transmembrane domains of putative the NH ₂ -terminal part of the protein. Since these cysteines are conserved in the LH, FSH and TSH receptors, disulfide bond formation may be crucial for the structural stability of the large extracellular domain of the glycoprotein hormone receptor. unknown.

The domain believed to contain the transmembrane region is approximately 90 with the rat and porcine LH / hCG receptors.
% Identity and 70 with TSH and FSH receptors
It has a% identity. Serine and threonine are frequently found in putative intracellular domains with three sites that are likely to undergo phosphorylation by protein kinase C (FIG. 1). Phosphorylation by receptor-specific kinases plays a role in agonist-specific elimination of adrenergic receptors from G-proteins, so that at least one of these sites is phosphorylated and thereby the LH / hCG receptor. It is important to know if it will cause any functional changes in.

In the present invention, in addition to a clone having a large open reading frame, a clone encoding a shorter protein was obtained. The large clones are from 1 to 699 in FIG. 1, and the truncated ones are from 227 to
The part surrounded by the square 289 is missing. This format suggests that the cleavage machinery required to complete the mRNA is selective. These results are very similar to the data for porcine LH / hCG receptor. The role of this truncated receptor is not well understood and this LH
It is also unknown whether the / hCG receptor is physiologically active as a monomer or oligomer. In humans, this TSH receptor can be the target of autoimmune reactions that lead to hyper- or hypostimulation of the thyroid by autoantibodies in Graves' disease and idiopathic myxedema. Thus, to better understand ovarian physiology as well as contribute to the diagnosis and management of ovarian diseases,
It is necessary to isolate the human LH / hCG receptor and to characterize it.

FIG. 2 shows the amino acid sequences of the novel human luteinizing hormone-human chorionic gonadotropin receptor protein obtained by the present invention, the rat and porcine luteinizing hormone-human chorionic gonadotropin receptor protein and the similar action. The amino acid sequences of the FSH and TSH receptors having the same amino acid sequence as those of the human luteinizing hormone-human chorionic gonadotropin receptor protein of the present invention are shown as ".", And human LH / hC
The amino acid portion that differs from the G receptor is represented by a one-letter expression. CONSENSUS in this figure shows the amino acids common to all of the glycoproteins listed in this figure.
Since ENSUS is provided, the missing part "-" is provided in the formula. Therefore, the numbers representing amino acids are counted excluding this missing part.

Regarding the DNA sequence, the DNA encoding the human LH / hCG receptor of the present invention contains the nucleotide sequence shown in FIG. 1 or a part thereof.

The cDNA encoding the human LH / hCG receptor of the present invention may be any one as long as it contains a nucleotide sequence encoding the amino acid sequence of the human LH / hCG receptor. The DNA containing the nucleotide sequence shown in FIG. 1 or a part of the DNA is preferable. The nucleotide sequence shown in FIG. 1 is an example of the cDNA sequence encoding the human LH / hCG receptor obtained in the present invention.

The expression vector containing the cDNA having the nucleotide sequence encoding the human LH / hCG receptor in the method of the present invention is, for example, (i) human messenger RNA (mRNA) isolated from human LH / hCG receptor producing cells. (Ii) single-stranded complementary DNA (cDN
A) and then double-stranded DNA is synthesized, (iii) the complementary DNA is incorporated into a phage or plasmid, (iv)
A host is transformed with the obtained recombinant phage or plasmid, and (v) the obtained transformant is cultivated, and then the transformant is subjected to an appropriate method, for example, a DNA probe encoding a part of rat LH / hCG receptor. A phage or plasmid containing the target DNA by hybridization with or by an immunoassay method using an anti-LH / hCG receptor antibody, and (vi) cutting out the target cloned DNA from the recombinant DNA, (Vii) It can be produced by ligating the cloned DNA or a part thereof downstream of a promoter in an expression vector.

MR encoding the human LH / hCG receptor
NA is a variety of human LH / hCG receptor producing cells such as testicular Leydig cells, ovarian capsular cells, granulosa cells,
It can be obtained from germ cells such as luteal cells and stromal cells. RNA from human LH / hCG receptor producing cells
The guanidine thiocyanate method [(JM. Chirgwin, et al., Bio-chemistry, 18 , 5294 (1979)] and the like can be mentioned as a method for preparing the above. Using a reverse transcriptase as a template, the method of Okayama (H. Okayama) et al. (Molecular and Cellular Biology) 2, 161 (1982) and ibid . 280 (1983)], and the resulting cDNA is incorporated into a plasmid. Examples of the plasmid into which the cDNA is incorporated include pBR322 derived from Escherichia coli.
[Gene, 2, 95 (1977)], pBR325 [gene, 4, 12
1 (1978)], pUC12 [Gene, 19 ,, 259 (1982)], pUC13
[Gene, 19, 259 (1982)], pUB110 derived from Bacillus subtilis [Biochemical and Biophysical Research Comm
unication), 112, 678 (1983)] and the like, but any of them can be used as long as they can be replicated and propagated in the host. In addition, examples of phage vectors that incorporate cDNA include λgt11
(Young, R., and Davis, R.,)
Proceedings of the National Academy of Sciences of the USA (P
roc. Natl. Acad. Sci., USA), 80 , 1194 (1983)] and the like, but other ones can be used as long as they can be propagated in the host.

The method for incorporating into a plasmid is, for example, T. Maniatis et al., Molecular Cloning Cold Spring Harbor Laboratory.
or La-boratory), page 239 (1982), and the like. In addition, as a method of incorporating cDNA into a phage vector, for example, the method of Hyunh, TV, et al. [DNA Cloning, A Practical Approach] 1 ,
49 (1985)] and the like. The plasmid thus obtained can be used in a suitable host, such as Escherichia
It is introduced into the genus richia and the genus Bacillus. As an example of the above-mentioned Escherichia bacterium, Escherichia coli K12DH1 [Proc. Natl. Acad. Sci. USA 60, 160 (1968)] , M
103 (Nucleic Acids Research, (Nucleic Acids
Research), 9, 309 (1981)], JA221 [Journal of Molecular Biology (Journal of Molecular
Biology)], 120, 517 (1978)], HB101 [Journal
Of Molecular Biology 41, 459 (1969)], C6
00 [Genetics, 39, 440 (1954)] and the like.

Examples of the bacterium belonging to the genus Bacillus include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry 95, 87 (198).
4)] and the like. As a method for transforming a host with a plasmid, for example, T. Maniatis (T. Mann
iatis) et al., Molecular Cloning (Molecular Clo
ning), Cold Spring Harbor Laboratory
(Cold Spring Harbor Laboratory), page 249 (1982), the calcium chloride method or the calcium chloride / rubidium chloride method. When a phage vector is used, it can be introduced into, for example, grown Escherichia coli by the in vitro packaging method. The human LH / hCG receptor / cDNA library containing the human LH / hCG receptor cDNA can be obtained by the above-mentioned method or the like, or can be purchased as a commercial product. The cDNA library can be obtained from Clontech Laboratories, Inc., USA.

Human LH / h from human DNA library
As a method for cloning the CG receptor cDNA,
For example, phage vector λcharon28A and rat LH / h
Plaque hybridization method using CG receptor cDNA as a probe [T.
niatis) et al., Molecular Cloning (Molecular Clo
Ning) Cold Spring Harbor La-boratory, (1982)]. Human LH cloned in this way
/ HCG receptor cDNA is a plasmid if necessary, such as pBR322, pUC12, pUC13, pUC18, pUC19, pUC118, pUC
Human LH / hCG receptor cDNA can be obtained by subcloning into 119 or the like.

The nucleotide sequence of the cDNA thus obtained is shown in, for example, Maxam-Gilbert.
Law [Maxam, AM and Gilbert, w., Proc. Natl. Acad., Proceedings of the National Academy of Sciences of the USA.
Sci., USA), 74 , 560 (1977)] or dideoxy method [Messing, J. et al., Nucleic Acids Research.
(Nucleic Acids Research) 9, 309 (1981)] and compared with known amino acid sequences, human LH / hCG
Confirm the presence of receptor cDNA. As described above,
A cDNA encoding the human LH / hCG receptor protein is obtained. Human LH / hCG obtained in Example 1 described later
The nucleotide sequence of the cDNA encoding the receptor protein determined by the dideoxy method and the amino acid sequence found from the nucleotide sequence are shown in FIG.

Human L cloned as described above
The cDNA encoding the H / hCG receptor protein can be used as it is, or if desired, digested with a restriction enzyme before use. An expression vector can be obtained by cutting out a region to be expressed from the cloned cDNA and ligating it downstream of a promoter in a vehicle (vector) suitable for expression. The cDNA has ATG as a translation initiation codon at its 5'end, and TAA, TGA or TAG as a translation stop codon at its 3'end.
May have. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adaptor. Furthermore, in order to express the DNA, a promoter is connected to the upstream thereof. As a vector, a plasmid derived from the above E. coli (eg, pBR32
2, pBR325, pUC12, pUC13), and a Bacillus subtilis-derived plasmid (eg, pUB110, pTP5, pC1)
94), yeast-derived plasmid (eg, pSH19, pSH
15), or bacteriophages such as λ phage and retroviruses, animal viruses such as vaccinia virus, and the like.

The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. When the host for transformation is a genus Escherichia, the trp promoter, lac promoter, recA promoter,
λPL promoter, lpp promoter, etc., when the host is Bacillus, SPO1 promoter,
When the host is yeast such as SPO2 promoter and penP promoter, PHO5 promoter and PGK
A promoter, GAP promoter, ADH promoter and the like are preferable. Particularly, it is preferable that the host is Escherichia and the promoter is the trp promoter or the λPL promoter. When the host is an animal cell, SV40-derived promoter, retrovirus promoter, metallothionein promoter, heat shock promoter and the like can be used. The use of enhancers for expression is also effective.

Human LH / hC constructed in this way
A transformant is produced using a vector containing a cDNA encoding the mature peptide of the G receptor protein. Examples of the host include Escherichia, Bacillus, yeast, animal cells and the like. Specific examples of the Escherichia genus and Bacillus genus include the same ones as described above. Examples of the yeast include Saccaromyces cerevis
iae) AH22, AH22R ~, NA87-11A, DKD
-5D etc. are mentioned. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO, mouse L cells, human FL cells and the like.

To transform the above-mentioned Escherichia bacterium, for example, Procedure of the National
Academy of Science (Proc.Natl.Acad.Sci.U
SA), 69 , 2110 (1972) and Jean, 17 , 107 (1982). For transformation of Bacillus, for example, Molecular & General Genetics, 168 ,
111 (1979) and the like. For transforming yeast, for example, the procedure of
The National Academy of Science (Pro
c. Natl. Acad. Sci. USA), 75 , 1929 (1978). Transformation of animal cells is performed, for example, according to the method described in Virology 52 , 456 (1973). Thus, human LH / hCG
A transformant transformed with the expression vector containing the cDNA encoding the receptor is obtained.

When a transformant whose host is a bacterium of the genus Escherichia or a bacterium of the genus Bacillus is cultivated, a liquid medium is suitable as a medium used for the culturing, and among them, a liquid medium is necessary and necessary for the growth of the transformant. It contains a carbon source, a nitrogen source, an inorganic substance and the like. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. Examples of organic substances and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast, vitamins, growth promoting factors and the like may be added.
The pH of the medium is preferably about 5-8. As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics], Four
31-433, Cold Spring Harbor Laboratory, New York 19
72] is preferred. If necessary, a drug such as 3β-indolylacrylic acid can be added to the promoter so as to work efficiently. When the host is Escherichia, the culture is usually performed at about 15 to 43 ° C for about 3 to 24
It is carried out for a time, and if necessary, aeration and stirring can be added. When the host is Bacillus, the culture is usually about 30-4.
It may be performed at 0 ° C. for about 6 to 24 hours, and aeration and stirring may be added if necessary. When culturing a transformant whose host is yeast, examples of the medium include Burkholder (B
urkholder) Minimal medium [Bostian, KL et al., “Procedure of the National Academy of Science”
(Proc.Natl.Acad.Sci.USA) 77 , 4505 (1980)]. The pH of the medium is preferably adjusted to about 5-8. Culturing is usually carried out at about 20 ° C to 35 ° C for about 24 to 72 hours, and aeration and stirring are added if necessary.

When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing about 5 to 20% fetal bovine serum [Science 122 , 501 (19).
52)], DMEM medium [Viro-logy, 8 , 396
(1959)], RPMI1640 medium [Journal of
The American Medical Association (The
Jounal of the American Medical Association) 199 , 51
9 (1967)], 199 medium [Pro-ceeding of the Society for the Biological Me
dicine) 73 , 1 (1950)] and the like. pH is about 6
It is preferably -8. Cultivation is usually about 30-40 ° C
For about 15 to 60 hours, and aeration and stirring are added if necessary.

The human LH / hCG receptor protein can be separated and purified from the above culture by the following method, for example. When the human LH / hCG receptor protein is extracted from the cultured cells or cells, the cells or cells are collected by a known method after culturing, and the cells or cells are suspended in an appropriate buffer solution and then subjected to ultrasonic wave, lysozyme and / or After disrupting the cells or cells by freeze-thawing or the like, a method of obtaining a crude extract of the mature peptide of BMP by centrifugation or filtration can be appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. Human L in culture
When the H / hCG receptor protein is secreted, after the completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected. The culture supernatant thus obtained or the human LH / hCG receptor contained in the extract can be subjected to an appropriate combination of separation / purification methods known per se. Known separation and purification methods for these are methods utilizing solubility such as salting out and solvent precipitation,
Dialysis method, ultrafiltration method, gel filtration method, SDS-polyacrylamide gel electrophoresis method mainly utilizing difference in molecular weight, ion exchange chromatography utilizing difference in charge, affinity chromatography, etc. And a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography and a method utilizing a difference in isoelectric point such as isoelectric focusing. Also, human LH / hCG receptor complementation D
A method using an antibody against the fusion protein expressed by fusing NA with E. coli-derived DNAlacZ as an immunoaffinity column is also conceivable. Human L thus produced
The H / hCG receptor protein can be measured by an enzyme immunoassay using a specific antibody.

[0024]

[Effects] A large amount of human LH / hCG receptor can be produced in cells or cells infected with DNA or transformed with the cDNA of the present invention, and human LH / hCG receptor protein production can be advantageously induced. .. The human LH / hCG receptor protein produced here is used for research of ovarian physiology, provision of antibodies to the receptor, diagnosis and management of ovarian and testicular diseases such as ovulation abnormality and sperm reduction, and development of contraceptives. It is connected and, in humans, this TSH receptor may be the target of the autoimmune reaction that leads to hyperthyroidism or hypostimulation of the thyroid by autoantibodies in Graves' disease and idiopathic myxedema, so May inhibit the LH action in vivo or may cause overstimulation in place of LH, resulting in a pathological condition in the human reproductive system. As a method for detecting this anti-receptor antibody, any of the above It is possible to produce a receptor by such a method, perform labeling, and examine whether or not a substance (antibody) that binds to this is present in the living body.
In addition to this, a part or all of this receptor cDNA is expressed to produce an antibody against this, and this antibody produces LH.
Inhibition of the action, that is, application as a contraceptive is considered possible.

The human LH / hCG receptor protein of the present invention is orally or parenterally administered to a mammal (eg, human, rabbit, rat, mouse, etc.) by itself or as a pharmaceutical composition in a suitable dosage form. can do. The pharmaceutical composition used for the above administration is the human LH of the present invention as described above.
/ HCG receptor protein and pharmacologically acceptable carrier,
It includes a diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration.

That is, for example, as a composition for oral administration, solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (Including soft capsules), syrups, emulsions, suspensions and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient usually used in the field of formulation. Examples of carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate and the like.

Examples of the composition for parenteral administration include injections and suppositories. The injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections and drip injections. Includes dosage forms. Such an injection is prepared by a method known per se, that is, by dissolving, suspending or emulsifying the human LH / hCG receptor protein of the present invention in a sterile aqueous or oily liquid usually used for injections. Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants, and suitable solubilizing agent,
For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, polysorbate 80, H
CO-50 (polyoxyethylene (50mol) adduct of hydrogenat
ed castor oil)] etc. Examples of the oily liquid include sesame oil and soybean oil, and solubilizing agents such as benzyl benzoate and benzyl alcohol may be used in combination. The prepared injection solution is usually filled in a suitable ampoule. The suppository used for rectal administration is human LH of the present invention.
/ HCG receptor protein is prepared by mixing with a conventional suppository base.

As described above, cloning of the cDNA encoding the human LH / hCG receptor protein, preparation of the human LH / hCG receptor protein expression vector, production of a transformant therewith, and human LH using the transformant. / HCG
The production of the receptor protein and its usefulness are described in detail.

In the specification and drawings of the present invention, when an abbreviation is used for a base or amino acid, IUPAC-IU
It is based on the abbreviations by B Commision on Biochemical Nomenclature or the abbreviations commonly used in this field, and examples thereof are given below. When amino acids may have optical isomers, the L-form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphoric acid dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly or G: Glycine Ala or A: Alanine Val or V: Valine Leu or L: Leucine Ile or I: Isoleucine Ser or S: Serine Thr or T: Threonine Cys or C: Cysteine Met or M: Methionine Glu or E: Glutamic acid Asp Is D: aspartic acid Lys or K: lysine Arg or R: arginine His or H: histidine Phe or F: phenylalanine Tyr or Y: tyrosine Trp or W: tryptophan Pro or P: proline Asn or N: asparagine Gln or Q: Glutamine In the human luteinizing hormone-human chorionic gonadotropin receptor protein of the present invention, a part of its amino acid sequence may be modified (addition, removal, substitution with other amino acids, etc.).

[0030]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.
The transformant Escherichia coli JM109 / pUC18 obtained in Example 1 described below was accepted by the Ministry of International Trade and Industry, Institute of Industrial Science and Technology (FRI) under the contract number FERM BP on October 9, 1990. -3127
The microorganism was deposited at the Fermentation Research Institute (IFO) with an accession number IFO from October 11, 1990.
Deposited as 15096.

The transformant Escherichia coli DH1 / pHLH obtained in Example 2 described later.
R (UEX2) and Escherichia coli
a coli) JM109 / pHLHR (GEX-3X) was accepted by the Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Microorganism Research Institute (FRI) on August 29, 1991, under the contract number FERM BP-3.
545 and FERM BP-3544, and the microorganisms have been deposited at the Fermentation Research Institute (IFO) since August 30, 1991 with deposit numbers IFO 1521.
9, deposited as IFO 15218.

[0032]

Example 1 1) Preparation of cDNA library derived from human ovary Total R from human ovary by guanidine thiocyanate method
After extracting NA, mRNA was purified using an oligo (dT) cellulose column (Pharmacia, type 7). c
Purified mRN using DNA synthesis kit (Pharmacia)
A cDNA was synthesized from about 2 μg of A. After blunting the ends of this cDNA with T4 DNA polymerase, EcoRI
An adapter was added. This cDNA was ligated to the λgt10 vector and subjected to in vitro packaging using Gigapack Gold (Stratagene). This library contained 1 x 10 ⁶ independent recombinants and was propagated.

2) Purification of probe A rat LH / hCG receptor was cloned from a cDNA library prepared from rat ovary and inserted into a λZaPII vector (Stratagene) in the same manner as described above to obtain clone Zap3-5. -1 (2.8 kb) had been isolated.
sham), [α- ³² P] -dNTP (deoxynucleoside tr
iphosphate) was used for labeling.

3) Screening 5 × 10 ⁴ pfu (plaque forming unit)
Min λgt10 cDNA library phage solution and 5
Mix with 00μl of C600hfl (overnight culture), 37 ℃, 15
After incubating for 2 minutes, add 8 ml of 0.75% agarose (Nippon Gene) LB and 1.5% agar LB plate (15 cm
Seeds). A nitrocellulose filter (Hybond-N) (Amersham) was placed on the plaque-formed plate to fix the DNA. Then filter 6 × S
SC (0.15M NaCl, 0.015M sodium citrate,
0.1% BSA (bovine serum albumin) in pH 7.0),
Polyvinylpyrrolidone, 5% with Ficoll 400 (Pharmacia)
65 ° C in a solution containing pyrophosphoric acid and 0.1% SDS at 1-
Prehybridized for 2 hours. For hybridization, a probe was added with a guideline of 200,000 cpm / ml. Wash with 6X SSC, 0.1% SDS at 42 ° C for 15 minutes, then 0.1
× 70 in SSC, 0.1% SDS at 65 ℃ for 10 minutes, -70
Autoradiography was performed at ° C.

4) DNA Sequence Analysis Several clones were identified, the longest of which was selected for sequence analysis. This clone is pUC
18 (Takara), and transforming Escherichia coli JM109 with this plasmid, transformant Escherichia coli JM109 / pUC1
Got 8. This transformant was further stepwise scraped by exonuclease digestion to prepare single-stranded DNA from large to short ones. Sequence analysis was performed by the dideoxy chain end method using the 7DEAZA sequencing kit. LKB2010 Macrophor Sequencing Syst
Electrophoresis using em, data analysis is SDC G
I used enetyx. FIG. 1 shows the nucleotide sequence of this human LH / hCG receptor protein DNA and the amino acid sequence deduced therefrom. The DNA obtained here
Is the addition of eight DNAs from -8 to -1 at the N-terminal of SEQ ID NO: 1.

[0036]

Example 2 Expression of Human LH / hCG Receptor (HLHR) Protein (1) The HLHR cDNA clone obtained in Example 1 was used to encode the extracellular portion of this HLHR.
The 0 bp EcoRI-Xba fragment was added to the Bam of pUEX2.
Lac by inserting into the HI site and cloning
A Z-HLHR fusion gene was obtained. This lacZ-HLH
E. coli using the R fusion gene. E. coli DH1 was transformed with the transformant E. coli. coli DH1 / pHLHR
(UEX2) (FERM BP-3545) was obtained. The transformant was cultured overnight in LB at 30 ° C.
0 μl was seeded in 5 ml of LB medium. After incubating at 30 ° C. for 2 hours with air agitation and further at 42 ° C. for 2 hours, the cells were pelleted by centrifugation. The pelleted cells were immediately lysed in SDS-PAGE buffer and separated by 5% SDS-PAGE. At the same time, Escherichia coli in which only vector pUEX2 was incorporated was also 5% SDS-PA.
Attached to GE. After electrophoresis, the gel was stained with Coomassie blue. The result is shown in FIG. Lane 1 is a molecular weight marker, lane 2 is E. coli in which only the vector pUEX2 is incorporated, and lane 3 is that of the transformant of the present invention. Band 110Kd incorporating vector only
It was confirmed that “a” disappeared and a new protein of 159 Kda could be detected. The result of this electrophoresis and the result of the base sequence analysis show the expression of the HLHR protein.

(2) HLHR cD obtained in Example 1
Using the NA clone, a 1400 bp EcoRI-Xba fragment encoding the extracellular portion of this HLHR was PGE.
The glutathione S-transferase (GST) -HLHR fusion gene was obtained by inserting and cloning into the BamHI site of X-3X (Pharmacia). Using this GST-HLHR fusion gene, E. coli E. co
li JM109 was transformed into transformant E. col
i JM109 / pHLHR (GEX-3X) (FER
M BP-3544) was obtained. The transformants were cultivated overnight, this culture was diluted 1:10 with 500 ml of fresh medium and cultivated at 37 ° C. for 1 hour, then IPTG was added to 0.
Added until 1 mM. After further culturing for 7 hours, the cells were pelleted by centrifugation. The pelleted cells were immediately lysed in SDS-PAGE buffer and 10%
Separated by SDS-PAGE and simultaneously vector pGEX
E. coli containing only -3X is 10% SDS-PAG
Attached to E. After electrophoresis, the gel was stained with Coomassie blue. The result is shown in FIG. Lane 1 is a molecular weight marker, lane 2 is E. coli incorporating only the vector pGEX-3X, and lane 3 is that of the transformant of the present invention. Band 26Kd incorporating vector only
It was confirmed that a disappeared, and a new protein of 75 Kda could be detected. The result of this electrophoresis and the result of the base sequence analysis show the expression of the HLHR protein.

(3) HLHR cD obtained in Example 1
The entire coding region and additional flanking regions (2995b) on the EcoRI fragment (2995bp) of the NA clone.
The expression vector pCHLHR was constructed by inserting p) into the pCDNA 1 vector. Human kidney 293
The cells (ATTC CRL 1573) were kept in Dulbecco's modified Eagle medium containing 10% fetal bovine serum in humidified air containing 5% carbon dioxide. These cells were transiently (transi) transfected with calcium phosphate using pCHLHR, which is an expression vector encoding the full-length human LH / hCG receptor.
ently) transfected. Responsiveness to hCG was tested by looking at elevated cAMP levels on these cells. The result is shown in FIG. In FIG. 5, points indicate average values, and lines indicate data ranges. The intracellular cAMP level increased in accordance with the hCG hormone concentration, indicating that the transformed cells express the HLHR protein, which reacts with hCG to produce cAMP.

[0039]

[0040]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 2987 Sequence Type: Nucleic Acid Number of Strands: Double-Stranded Topology: Linear Sequence Type: cDNA to genomic DNA Origin: Human Ovary Sequence: ATG AAG CAG CGG TTC TCG GCG CTG CAG CTG CTG AAG CTG CTG CTG CTG 48 Met Lys Gln Arg Phe Ser Ala Leu Gln Leu Leu Lys Leu Leu Leu Leu 1 5 10 15 CTG CAG CCG CCG CTG CCA CGA GCG CTG CGC GAG GCG CTC TGC CCT GAG 96 Leu Gln Pro Pro Leu Pro Arg Ala Leu Arg Glu Ala Leu Cys Pro Glu 20 25 30 CCC TGC AAC TGC GTG CCC GAC GGC GCC CTG CGC TGC CCC GGC CCC ACG 144 Pro Cys Asn Cys Val Pro Asp Gly Ala Leu Arg Cys Pro Gly Pro Thr 35 40 45 GCC GGT CTC ACT CGA CTA TCA CTT GCC TAC CTC CCT GTC AAA GTG ATC 192 Ala Gly Leu Thr Arg Leu Ser Leu Ala Tyr Leu Pro Val Lys Val Ile 50 55 60 CCA TCT CAA GCT TTC AGA GGA CTT AAT GAG GTC ATA AAA ATT GAA ATC 240 Pro Ser Gln Ala Phe Arg Gly Leu Asn Glu Val Ile Lys Ile Glu Ile 65 70 75 80 TCT CAG ATT GAT TCC CTG GAA AGG ATA GAA GCT AAT GCC TTT GAC AAC 288 Ser Gln Ile Asp Ser Leu Glu Arg Ile Glu Ala Asn Ala Phe Asp Asn 85 90 95 CTC CTC AAT TTG TCT GAA ATA CTG ATC CAG AAC ACC AAA AAT CTG AGA 336 Leu Leu Asn Leu Ser Glu Ile Leu Ile Gln Asn Thr Lys Asn Leu Arg 100 105 110 TAC ATT GAG CCC GGA GCA TTT ATA AAT CTT CCC GGA TTA AAA TAC TTG 384 Tyr Ile Glu Pro Gly Ala Phe Ile Asn Leu Pro Gly Leu Lys Tyr Leu 115 120 125 AGC ATC TGT AAC ACA GGC ATC AGA AAG TTT CCA GAT GTT ACG AAG GTC 432 Ser Ile Cys Asn Thr Gly Ile Arg Lys Phe Pro Asp Val Thr Lys Val 130 135 140 TTC TCC TCT GAA TCA AAT TTC ATT CTG GAA ATT TGT GAT AAC TTA CAC 480 Phe Ser Ser Glu Ser Asn Phe Ile Leu Glu Ile Cys Asp Asn Leu His 145 150 155 160 ATA ACC ACC ATA CCA GGA AAT GCT TTT CAA GGG ATG AAT AAT GAA TCT 528 Ile Thr Thr Ile Pro Gly Asn Ala Phe Gln Gly Met Asn Asn Glu Ser 165 170 175 GTA ACA CTC AAA CTA TAT GGA AAT GGA TTT GAA GAA GTA CAA AGT CAT 576 Val Thr Leu Lys Leu Tyr Gly Asn Gly Phe Glu Glu Val Gln Ser His 180 185 190 GCA TTC AAT GGG ACG ACA CTG ACT TCA CTG GAG CTA AAG GAA AAC GTA 624 Ala Phe Asn Gly Thr Thr Leu Thr Ser Leu Glu Leu Ly s Glu Asn Val 195 200 205 CAT CTG GAG AAG ATG CAC AAT GGA GCC TTC CGT GGG GCC ACA GGG CCG 672 His Leu Glu Lys Met His Asn Gly Ala Phe Arg Gly Ala Thr Gly Pro 210 215 220 AAA ACC TTG GAT ATT TCT TCC ACC AAA TTG CAG GCC CTG CCG AGC TAT 720 Lys Thr Leu Asp Ile Ser Ser Thr Lys Leu Gln Ala Leu Pro Ser Tyr 225 230 235 240 GGC CTA GAG TCC ATT CAG AGG CTA ATT GCC ACG TCA TCC TAT TCT CTA 768 Gly Leu Glu Ser Ile Gln Arg Leu Ile Ala Thr Ser Ser Tyr Ser Leu 245 250 255 AAA AAA TTG CCA TCA AGA GAA ACA TTT GTC AAT CTC CTG GAG GCC ACG 816 Lys Lys Leu Pro Ser Arg Glu Thr Phe Val Asn Leu Leu Glu Ala Thr 260 265 270 TTG ACT TAC CCC AGC CAC TGC TGT GCT TTT AGA AAC TTG CCA ACA AAA 864 Leu Thr Tyr Pro Ser His Cys Cys Ala Phe Arg Asn Leu Pro Thr Lys 275 280 285 GAA CAG AAT TTT TCA CAT TCC ATT TCT GAA AAC TTT TCC AAA CAA TGT 912 Glu Gln Asn Phe Ser His Ser Ile Ser Glu Asn Phe Ser Lys Gln Cys 290 295 300 GAA AGC ACA GTA AGG AAA GTG AGT AAC AAA ACA CTT TAT TCT TCC ATG 960 Glu Ser Thr Val Arg Lys Val Ser Asn Ly s Thr Leu Tyr Ser Ser Met 305 310 315 320 CTT GCT GAG AGT GAA CTG AGT GGC TGG GAC TAT GAA TAT GGT TTC TGC 1008 Leu Ala Glu Ser Glu Leu Ser Gly Trp Asp Tyr Glu Tyr Gly Phe Cys 325 330 335 TTA CCC AAG ACA CCC CGA TGT GCT CCT GAA CCA GAT GCT TTT AAT CCC 1056 Leu Pro Lys Thr Pro Arg Cys Ala Pro Glu Pro Asp Ala Phe Asn Pro 340 345 350 TGT GAA GAC ATT ATG GGC TAT GAC TTC CTT AGG GTC CTG ATT TGG CTG 1104 Cys Glu Asp Ile Met Gly Tyr Asp Phe Leu Arg Val Leu Ile Trp Leu 355 360 365 ATT AAT ATT CTA GCC ATC ATG GGA AAC ATG ACT GTT CTT TTT GTT CTC 1152 Ile Asn Ile Leu Ala Ile Met Gly Asn Met Thr Val Leu Phe Val Leu 370 375 380 CTG ACA AGT CGT TAC AAA CTT ACA GTG CCT CGT TTT CTC ATG TGC AAT 1200 Leu Thr Ser Arg Tyr Lys Leu Thr Val Pro Arg Phe Leu Met Cys Asn 385 390 395 400 CTC TCC TTT GCA GAC TTT TGC ATG GGG CTC TAT CTG CTG CTC ATA GCC 1248 Leu Ser Phe Ala Asp Phe Cys Met Gly Leu Tyr Leu Leu Leu Ile Ala 405 410 415 TCA GTT GAT TCC CAA ACC AAG GGC CAG TAC TAT AAC CAT GCC ATA GAC 1296 Ser Val Asp Se r Gln Thr Lys Gly Gln Tyr Tyr Asn His Ala Ile Asp 420 425 430 TGG CAG ACA GGG AGT GGG TGC AGC ACT GCT GGC TTT TTC ACT GTA TTC 1344 Trp Gln Thr Gly Ser Gly Cys Ser Thr Ala Gly Phe Phe Thr Val Phe 435 440 445 GCA AGT GAA CTT TCT GTC TAC ACC CTC ACC GTC ATC ACT CTA GAA AGA 1392 Ala Ser Glu Leu Ser Val Tyr Thr Leu Thr Val Ile Thr Leu Glu Arg 450 455 460 TGG CAC ACC ATC ACC TAT GCT ATT CAC CTG GAC CAA AAG CTG CGA TTA 1440 Trp His Thr Ile Thr Tyr Ala Ile His Leu Asp Gln Lys Leu Arg Leu 465 470 475 480 AGA CAT GCC ATT CTG ATT ATG CTT GGA GGA TGG CTC TTT TCT TCT CTA 1488 Arg His Ala Ile Leu Ile Met Leu Gly Gly Trp Leu Phe Ser Ser Leu 485 490 495 ATT GCT ATG TTG CCC CTT GTC GGT GTC AGC AAT TAC ATG AAG GTC AGT 1536 Ile Ala Met Leu Pro Leu Val Gly Val Ser Asn Tyr Met Lys Val Ser 500 505 510 ATT TGC TTC CCC ATG GAT GTG GAA ACC ACT CTC TCA CAA GTC TAT ATA 1584 Ile Cys Phe Pro Met Asp Val Glu Thr Thr Leu Ser Gln Val Tyr Ile 515 520 525 TTA ACC ATC CTG ATT CTC AAT GTG GTG GCC TTC TTC ATA ATT TGT GCT 1632 Leu Thr Ile Leu Ile Leu Asn Val Val Ala Phe Phe Ile Ile Cys Ala 530 535 540 TGC TAC ATT AAA ATT TAT TTT GCA GTT CGA AAC CCA GAA TTA ATG GCT 1680 Cys Tyr Ile Lys Ile Tyr Phe Ala Val Arg Asn Pro Glu Leu Met Ala 545 550 555 560 ACC AAT AAA GAT ACA AAG ATT GCT AAG AAA ATG GCA ATC CTC ATC TTC 1728 Thr Asn Lys Asp Thr Lys Ile Ala Lys Lys Met Ala Ile Leu Ile Phe 565 570 575 ACC GAT TTC ACC TGC ATG GCA CCT ATC TCT TTT TTT GCC ATC TCA GCT 1776 Thr Asp Phe Thr Cys Met Ala Pro Ile Ser Phe Phe Ala Ile Ser Ala 580 585 590 GCC TTC AAA GTA CCT CTT ATC ACA GTA ACC AAC TCT AAA GTT TTA CTG 1824 Ala Phe Lys Val Pro Leu Ile Thr Val Thr Asn Ser Lys Val Leu Leu 595 600 605 GTT CTT TTT TAT CCC ATC AAT TCT TGT GCC AAT CCA TTT CTG TAT GCA 1872 Val Leu Phe Tyr Pro Ile Asn Ser Cys Ala Asn Pro Phe Leu Tyr Ala 610 615 620 ATA TTC ACT AAG ACA TTC CAA AGA GAT TTC TTT CTT TTG CTG AGC AAA 1920 Ile Phe Thr Lys Thr Phe Gln Arg Asp Phe Phe Leu Leu Leu Ser Lys 625 630 635 640 TTT GGC TGC TGT AAA CGT CGG GCT GAA CTT T AT AGA AGG AAA GAT TTT 1968 Phe Gly Cys Cys Lys Arg Arg Ala Glu Leu Tyr Arg Arg Lys Asp Phe 645 650 655 TCA GCT TAC ACC TCC AAC TGC AAA AAT GGC TTC ACT GGA TCA AAT AAG 2016 Ser Ala Tyr Thr Ser Asn Cys Lys Asn Gly Phe Thr Gly Ser Asn Lys 660 665 670 CCT TCT CAA TCC ACC TTG AAG TTG TCC ACA TTG CAC TGT CAA GGT ACA 2064 Pro Ser Gln Ser Thr Leu Lys Leu Ser Thr Leu His Cys Gln Gly Thr 675 680 685 GCT CTC CTA GAC AAG ACT CGC TAC ACA GAG TGT TAACTGTTAC ATCAGTAACT 2117 Ala Leu Leu Asp Lys Thr Arg Tyr Thr Glu Cys 690 695 699 GCATTATTGA ATTGTTCTTA AACCTGTAAA AAAAAATTAC CTGTACCAGT AATTTTAACA 2177 TAAAGGGTTG GATTTAGGAA ATTATTTATT TTTAGGTACA TTAGGCAAGA GACCTCTACC 2237 TAGTAGAAAG TGTAGTCTAT GACCACTGCC ACACGTAAAA ACTATTTGTC ATTGTTACAT 2297 GGCATAAATA TGAAGTTGAG AGTGTTTAGA AATTTTTATA GAAATTTTGA CACAGTAATT 2357 TTGTTTGATG AATCTTTTAA AAAACAGAGG AGGTATTTTG CATATCTTTT TTTCATTTTC 2417 GTAATTTGTA TTGCATTCTA TAAAAATATT AGTTCATAAC AGATCAGAAA TTTAAAATAATA 2477 GGGGCTTTC CCTAGAGAC ACTT T TCAATTCGGT 2537 ACGCACTAGC CACATGTGGC TAAATTAAAA TTAAATAAAA TGAGAAATGT AGTTTCTCAG 2597 TTGCACTACG TTTCAAGTTC TCAATGGCTA CGTCAAGTTC TCAATGGCTA CGTGTGACTA 2657 GTGCTTACCA TACTGGACAG CACAGACACA GAATATTTTC ATCACCACAG AAAGTTCTAT 2717 CTGTTCTATT ATAGAGACTT TTATGTATGC CCTATCTGGA TTCTACTTAT TTATAATTTA 2777 AGGTAAACAT CTGAAAGCAC ATTTCAGCCT ATTTGCTTAG TGAAACATTA AGCTGTAGAC 2837 TGTAAACTCC TCGTGAGTAG GAACCCTGTC TCAGTGCATT TTGTTTTCCT GCTTCCTACC 2897 TCAAGATCTT GGCAATGGTA CACTACAAAT GTGCTGAGTT AGAATTACTC TGAAGTTATG 2957 AAACATATAA TGAAAACAAT TTTTCCGGCC 2987

[Brief description of drawings]

FIG. 1 is a view showing the nucleotide sequence of human LH / hCG receptor protein DNA and the amino acid sequence deduced therefrom.

FIG. 2 is a diagram showing the comparison of the amino acid sequence of human LH / hCG receptor protein with the amino acid sequences of other known LH / hCG receptor proteins or proteins having a similar action.

FIG. 3 is a schematic diagram showing the result of SDS-PAGE showing that the human LH / hCG receptor protein was expressed in Example 2.

FIG. 4 is a schematic diagram showing the results of SDS-PAGE showing that the human LH / hCG receptor protein was expressed in Example 2.

FIG. 5 is a graph showing the reactivity of the human LH / hCG receptor protein obtained in Example 3 with hCG.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] September 10, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Detailed explanation of the invention

[Correction method] Change

[Correction content]

In the present invention, in addition to a clone having a large open reading frame, a clone encoding a shorter protein was obtained. Large clone those 1-699 amino acid sequence of Figure 1, and in which truncated the 227-289 th amino acid sequence (a portion enclosed by a square) is missing. This format suggests that the cleavage machinery required to complete the mRNA is selective. These results are very similar to the data for porcine LH / hCG receptor. The role of this truncated receptor is not well understood, and it is not known whether this LH / hCG receptor is physiologically active as a monomer or an oligomer. In person, this TSH
Receptors can be the target of autoimmune reactions that lead to hyper- or hypostimulation of the thyroid by autoantibodies in Graves' disease and idiopathic myxedema. Thus, it is necessary to isolate and characterize the human LH / hCG receptor in order to better understand ovarian physiology as well as contribute to the diagnosis and management of ovarian disease.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12P 21/02 C 8214-4B // (C12N 1/21 C12R 1:19) C12R 1:19)

Claims (10)

[Claims] 1. A human luteinizing hormone-human chorionic gonadotropin receptor protein. 2. The protein according to claim 1, which has the amino acid sequence of the 1st to 699th amino acids in SEQ ID NO: 1, or the amino acid sequence in which the 227th to 289th amino acids thereof are deleted, or a part thereof. 3. The protein according to claim 2, which has the amino acid sequence of the 26th to 699th amino acids in SEQ ID NO: 1 or the amino acid sequence in which the 227th to 289th amino acids thereof are deleted. 4. A DNA containing a cDNA encoding a human luteinizing hormone-human chorionic gonadotropin receptor protein. 5. The human luteinizing hormone-human chorionic gonadotropin receptor protein is 1-699 in SEQ ID NO: 1.
The DNA according to claim 4, which is a protein having the amino acid sequence at position 23, or the amino acid sequence at which the 227th to 289th amino acids are deleted, or a part thereof. 6. A cDNA encoding a human luteinizing hormone-human chorionic gonadotropin receptor protein, wherein the nucleotide sequence from 1 to 2987 in SEQ ID NO: 1 or the 679 to 866 nucleotide in the nucleotide sequence is deleted. The DNA according to claim 5, which contains a nucleotide sequence or a part thereof. 7. A cDNA encoding a human luteinizing hormone-human chorionic gonadotropin receptor protein has the nucleotide sequence of 76 to 2987 in SEQ ID NO: 1 or the 679 to 866 nucleotides thereof deleted. The DNA according to claim 6, which contains a nucleotide sequence. 8. A transformant retaining a DNA containing a cDNA encoding a human luteinizing hormone-human chorionic gonadotropin receptor protein. 9. A transformant carrying a DNA containing a cDNA encoding a human luteinizing hormone-human chorionic gonadotrophin receptor protein is cultured, and the human luteinizing hormone-human chorionic gonadotropin receptor is cultured in the culture. A method for producing a human luteinizing hormone-human chorionic gonadotropin receptor protein, which comprises producing and accumulating a protein and collecting the protein. 10. A pharmaceutical composition comprising an effective amount of human luteinizing hormone-human chorionic gonadotropin receptor protein and a pharmaceutically acceptable carrier or excipient.