JPH1072497A - Receptor protein specific to nervous cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new protein comprising a receptor protein containing a specific amino acid sequence and useful for inhibition of nervous cell differentiation of a receptor protein specific to nervous cell, especially that of an activin receptor and elucidation, etc., of mechanism of neurotrophic factor-like activity thereof. SOLUTION: This protein is a new receptor protein (salt) containing substantially same amino acid sequence as an amino acid sequence represented by the formula, especially a receptor protein specific to nervous cell, especially preferably a new activin receptor protein, and useful as means, etc., for elucidating function of a receptor specific to nervous cell, especially elucidating detailed mechanism of inhibition of nervous cell differentiation and neurotrophic factor- like activity, and the gene is used as a probe for diagnosing gene and a medicine for treating gene. The protein is obtained by screening a human genome DNA library by using a probe having a partial base sequence of the receptor protein, integrating the resultant gene into a vector and expressing the gene in a host cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a receptor protein having a specific amino acid sequence, particularly a receptor protein specific to nervous cells, particularly a novel activin receptor protein, and a D-protein containing a DNA region encoding the protein.
The present invention relates to NA, a method for producing the protein, a method for detecting a gene encoding the protein, and uses of the protein and DNA.

[0002]

2. Description of the Related Art Conventionally, many physiologically active substances have been isolated and identified, and their functions have been elucidated. It is known that some of them exhibit various activities in various organs or cells. Various biological activities in various organs or cells are usually expressed via a receptor to which the physiologically active substance binds. Whether the receptor is the same in all organs or cells, In many cases, the specificity has not been elucidated. Activin is a follicle-stimulating hormone (FSH) from the anterior pituitary gland
Was isolated as a regulator that promotes the secretion of Conventionally, it was thought that steroid hormones produced in the gonads were mainly responsible for the regulation of gonadotropin secretion from the pituitary gland. However, the discovery of activin and inhibin, which has an opposing action thereto, led to the hypothalamus-brain. It is of interest as a novel mechanism of pituitary-reproductive system hormone regulation. As the analysis of the bioactivity of activin progressed, this system was found to have various bioactivities other than regulating FSH secretion, such as the activity of inducing or inhibiting the differentiation of cells of the blood cell system and reproductive organs, and the activity of maintaining survival of nerve cells. However, there are many points where the detailed mechanism has not been elucidated yet. The activin receptor gene has already been cloned in humans, mice, rats, and the like, and two types of type I and type II are known from its structural analysis.
Furthermore, it was found that Type I and Type II have two types of subtypes, A and B, and that Type IIB is divided into four types by alternative splicing. These receptor proteins, which are thought to embody the various biological activities of activin, have different affinities for activin, but their expression is widespread, and organ or cell specificity is observed. Not known to be. In particular, activin is known to induce or inhibit cell differentiation, but it has not been known to date that its expression is significantly changed specifically in the differentiation or differentiation process. Mechanism has not yet been elucidated.

[0003]

DISCLOSURE OF THE INVENTION The present invention aims to elucidate the functions of nervous system cell-specific receptors, and in particular, to provide a detailed mechanism of activin receptors exhibiting various activities to inhibit nervous system cell differentiation and neurotrophic factor-like activities. As a means for elucidation, a method for isolating and detecting a novel activin receptor gene specific to the nervous system, including the novel receptor gene
An object of the present invention is to provide DNA, a method for producing a protein encoded by the novel receptor gene, and uses of the DNA and the protein.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that a synthetic DNA for efficiently isolating a cDNA encoding an activin receptor type IIA protein. Mouse embryonic tumor cells P19 derived from retinoic acid-induced neural differentiation using primers
oly (A) ⁺ RNA from reverse transcriptase polymerase chain reaction (Reverse-transcri
Using the ptase polymerase chain reaction (hereinafter abbreviated as RT-PCR) method, the cDNA fragment encoding the activin receptor type IIA protein was successfully amplified, and the analysis was proceeded. As a result, the present inventors have isolated DNA encoding a novel receptor protein. As a result of further analysis of this nucleotide sequence, the DNA
Encodes a novel subtype of the activin receptor type IIA protein containing eight amino acids represented by SEQ ID NO: 7, which is considered to be characteristic of the activin receptor. Further, the present inventors have conducted repeated studies, and have also found that the expression of the receptor gene is specifically expressed in cells and organs of the nervous system.

Further, the present inventors have found that the expression of the known activin receptor type IIA gene and the expression of the novel receptor type IIA gene can be easily distinguished and detected by RT-PCR using the synthetic DNA primer. Was found. More specifically, the present inventors, mouse embryonic tumor cells P19 cells derived from retinoic acid-induced neuronal differentiation poly (A)
^A primer selected from SEQ ID NO: 1 and the synthetic DNA shown in SEQ ID NO: 2 selected from the known mouse activin receptor type IIA gene (M65287: a number registered as data in GenBank) shown in FIGS. Used as
When a cDNA fragment was obtained by the RT-PCR method, a cDNA fragment having a long fragment length was amplified simultaneously with a fragment having the same fragment length as that expected from a known nucleotide sequence. From the results of analyzing the nucleotide sequence of this cDNA fragment, these cDNAs were obtained from the known activin receptor type IIA gene (M65
287) and a new splicing site of a known activin receptor type IIA gene (M65287).
Was found to encode the inserted receptor. Next, the present inventors
Mouse embryonic tumor cells P1 induced neural differentiation with retinoic acid
Create cDNA from 9 cell-derived poly (A) ⁺ RNA and convert this cDNA to λ
A cDNA library was prepared by insertion into ZAPII phage. Furthermore, a cDNA having a new 24 base pair sequence inserted into the splicing site obtained by the RT-PCR method is used as a probe to screen this library to completely encode a novel receptor protein of the present invention.
A fragment was obtained. As a result of analyzing the base sequence of this DNA fragment, as shown in SEQ ID NO: 4, except for the 24-base pair insertion found by the present inventors, the activin receptor type IIA gene (M65287 ) Matches its base sequence, and the deduced amino acid sequence is SEQ ID NO:
5 was found. (FIGS. 3 to 6
reference)

Further, the present inventors have extracted poly (A) ⁺ RNA from various organs of a mouse and performed RT-PCR by using the synthetic DNAs shown in SEQ ID NO: 1 and SEQ ID NO: 2 as primers. When the expression of the novel receptor was examined, it was found that the expression was specifically observed in the brain and all embryos as shown in FIG. In addition, cDNA fragments were obtained by RT-PCR using poly (A) ⁺ RNA derived from a cell line derived from human neuroblastoma and using the synthetic DNAs shown in SEQ ID NO: 1 and SEQ ID NO: 2 as primers. And frog embryo-derived poly (A) ⁺ RN
A cDNA fragment was obtained by RT-PCR using the synthetic DNA shown in SEQ ID NO: 9 and SEQ ID NO: 10 as a primer using A. As described above, the same fragment as the fragment expected from the known nucleotide sequence was obtained as described above. Along with the long fragment, a cDNA fragment with a long fragment length was amplified, and as a result of analyzing these base sequences, a similar 24-base pair sequence was inserted in humans and frogs. The same frog as the mouse was found to have the nucleotide sequence shown in SEQ ID NO: 6 in the frog. Also, when these nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 6 were translated into amino acid sequences, they all matched the amino acid sequence shown in SEQ ID NO: 7 and were found to be very well conserved across species. Was.

That is, the present invention relates to (1) a receptor protein having an amino acid sequence substantially identical to the amino acid sequence represented by the formula His Ala Phe His Ile Met Ile Glu or a salt thereof; A receptor protein according to the above (1), which is a neural cell-specific receptor protein; (3) a neural cell-specific receptor protein containing the amino acid sequence represented by SEQ ID NO: 7, or a salt thereof; : An activin receptor protein or a salt thereof comprising the amino acid sequence represented by: 7; (5) an activin receptor protein or a salt thereof comprising the amino acid sequence represented by SEQ ID NO: 5; (6) a receptor The receptor protein according to the above (2), wherein the protein is an activin receptor protein; (7) the protein is represented by the formula His Ala Phe His Ile Met Ile Glu It contains an amino acid sequence substantially identical to the amino acid sequence and has the formula X-Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn Trp Glu Arg Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Ile Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile Cys Ala Phe Trp Val Tyr Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gln His Ala Phe His Ile Met Ile Glu Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Leu Gly Leu Lys Pro Leu Gln Leu Leu Glu Val Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Leu Asn Glu Tyr Val Ala Val Lys Ile Phe Pro Ile Gln Asp Lys Gln Ser Trp Gln Asn Glu Tyr Glu Val Tyr Ser Leu Pro Gly Met Lys His Glu Asn Ile Leu Gln Phe Ile Gly Ala Glu Lys Arg Gly Thr Ser Val Asp Val Asp Leu Trp Leu Ile Thr Ala Phe His Glu Lys Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val Ser Trp Asn Glu Leu Cys His Ile Ala Glu Thr Met Ala Arg Gly Leu Ala Tyr Leu His Glu Asp Ile Pro Gly Leu Lys Asp Gly His Lys Pro Ala Ile Ser His Arg Asp Ile Lys Ser Lys Asn Val Leu Leu Lys Asn Asn Leu Thr Ala Cys Ile Ala Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala Gly Lys Ser Ala Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Ala Ser Arg Cys Thr Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Asp Met Gln Glu Val Val Val His Lys Lys Lys Arg Pro Val Leu Arg Asp Tyr Trp Gln Lys His Ala Gly Met Ala Met Leu Cys Glu Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Gly Glu Arg Ile Thr Gln Met Gln Arg Leu Thr Asn Ile Ile Thr Thr Glu Asp Ile Val Thr Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys Glu Ser Ser Leu (where X is protected Indicates Met or hydrogen which may be present. A receptor protein containing the amino acid sequence represented by the following formula or a partial sequence thereof, or a salt thereof: (8) Formula X-GlyAlaAlaAlaLysleuAlaPheAlaValPheLeuIle SerCys Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn Trp Glu Arg Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Ile Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile Cys Ala Phe Trp Val Tyr Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gln His Ala Phe His Ile Met Ile Glu Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Leu Gly Leu Lys Pro Leu Gln Leu Leu Glu Val Lys Ala Arg Gly Arg Phe Gly Cys Val Tr p Lys Ala Gln Leu Leu Asn Glu Tyr Val Ala Val Lys Ile Phe Pro Ile Gln Asp Lys Gln Ser Trp Gln Asn Glu Tyr Glu Val Tyr Ser Leu Pro Gly Met Lys His Glu Asn Ile Leu Gln Phe Ile Gly Ala Glu Lys Arg Gly Thr Ser Val Asp Val Asp Leu Trp Leu Ile Thr Ala Phe His Glu Lys Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val Ser Trp Asn Glu Leu Cys His Ile Ala Glu Thr Met Ala Arg Gly Leu Ala Tyr Leu His Glu Asp Ile Pro Gly Leu Lys Asp Gly His Lys Pro Ala Ile Ser His Arg Asp Ile Lys Ser Lys Asn Val Leu Leu Lys Asn Asn Leu Thr Ala Cys Ile Ala Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala Gly Lys Ser Ala Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Ala Ser Arg Cys Thr Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Asp Met Gln Glu Val Val Val His Lys Lys Lys Arg Pro Val Leu Arg Asp Tyr Trp Gln Lys His Ala Gly Met Ala Met Leu Cys Glu Th r Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Gly Glu Arg Ile Thr Gln Met Gln Arg Leu Thr Asn Ile Ile Thr Thr Glu Asp Ile Val Thr Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys Glu Ser Ser Leu wherein X represents optionally protected Met or hydrogen. In the amino acid sequence represented by the formula (I), one or more amino acids other than the amino acid sequence represented by the formula His Ala Phe His Ile Met Ile Glu may be deleted, added, inserted or substituted. ) The protein or a salt thereof, (9) Formula X-Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn Trp Glu Arg Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Ile Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile Cys Ala Phe Trp Val Tyr Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gln His Ala Phe His Ile Met Ile Glu Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Leu Gly Leu Lys Pro Leu Gln Leu Leu Glu Val Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Leu Asn Glu Tyr Val Ala Val Lys Ile Phe Pro Ile Gln Asp Lys Gln Ser Trp Gln Asn Glu Tyr Glu Val Tyr Ser Leu Pro Gly Met Lys His Glu Asn Ile Leu Gln Phe Ile Gly Ala Glu Lys Arg Gly Thr Ser Val Asp Val Asp Leu Trp Leu Ile Thr Ala Phe His Glu Lys Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val Ser Trp Asn Glu Leu Cys His Ile Ala Glu Thr Met Ala Arg Gly Leu Ala Tyr Leu His Glu Asp Ile Pro Gly Leu Lys Asp Gly His Lys Pro Ala Ile Ser His Arg Asp Ile Lys Ser Lys Asn Val Leu Leu Lys Asn Asn Leu Thr Ala Cys Ile Ala Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala Gly Lys Ser Ala Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Ala Ser Arg Cys Thr Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Asp Met Gln Glu Val Val Val His Lys Lys Lys Arg Pro Val Leu Arg Asp Tyr Trp Gln Lys His Ala Gly Met Ala Met Leu Cys Glu Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Gly Glu Arg Ile Thr Gln Met Gln Arg Leu Thr Asn Ile Ile Thr Thr Glu Asp Ile Val Thr Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys Glu Ser Ser Leu (where X is Indicates Met or hydrogen, which may be protected. [10] The protein or a salt thereof according to the above (1), which comprises an amino acid sequence represented by the formula [1], which comprises an amino acid sequence substantially identical to the amino acid sequence represented by the formula: , Activin or a protein capable of binding to a protein substantially the same as activin, or a salt thereof; (11) a DNA containing a base sequence encoding the protein of (1), (12) a DNA of (10) (13) a DNA according to the above (11) having a nucleotide sequence represented by SEQ ID NO: 4, (14) a DNA represented by the formula: X′GGA GCT GCT GCA AAG TTG GCG TTC GC
C GTC TTT CTT ATC TCT TGC TCT TCA GGT GCT ATA CTT GGC AGA TCA
GAA ACT CAG GAG TGT CTT TTC TTT AAT GCT AAT TGG GAA AGA GAC AGA
ACC AAC CAG ACT GGT GTT GAA CCT TGC TAT GGT GAT AAA GAT AAA CGG
CGA CAT TGT TTT GCT ACC TGG AAG AAT ATT TCT GGT TCC ATT GAA ATA
GTG AAG CAA GGT TGT TGG CTG GAT GAT ATC AAC TGC TAT GAC AGG ACT
GAT TGT ATA GAA AAA AAA GAC AGC CCT GAA GTG TAC TTT TGT TGC TGT
GAG GGC AAT ATG TGT AAT GAA AAG TTC TCT TAT TTT CCG GAG ATG GAA
GTC ACA CAG CCC ACT TCA AAT CCT GTT ACA CCG AAG CCA CCC TAT TAC
AAC ATT CTG CTG TAT TCC TTG GTA CCA CTA ATG TTA ATT GCA GGA ATT
GTC ATT TGT GCA TTT TGG GTG TAC AGA CAT CAC AAG ATG ATG GCC TAC CCT
CCT GTA CTT GTT CCT ACT CAA CAC GCC TTT CAT ATA ATG ATA GAG GAC
CCA GGA CCA CCC CCA CCT TCC CCA TTA CTA GGG TTG AAG CCA TTG CAG
CTG TTA GAA GTG AAA GCA AGG GGA AGA TTT GGT TGT GTC TGG AAA GCC
CAG TTG CTC AAT GAA TAT GTG GCT GTC AAA ATA TTT CCA ATA CAG GAC
AAA CAG TCC TGG CAG AAT GAA TAT GAA GTC TAT AGT CTA CCT GGA ATG
AAG CAT GAG AAC ATA CTA CAG TTC ATT GGT GCA GAG AAA AGA GGC ACC
AGT GTG GAT GTG GAC CTG TGG CTA ATC ACA GCA TTT CAT GAA AAG GGC
TCA CTG TCA GAC TTT CTT AAG GCT AAT GTG GTC TCT TGG AAT GAA CTT
TGT CAT ATT GCA GAA ACC ATG GCT AGA GGA TTG GCA TAT TTA CAT GAG
GAT ATA CCT GGC TTA AAA GAT GGC CAC AAG CCT GCA ATC TCT CAC AGG
GAC ATC AAA AGT AAA AAT GTG CTG CTG TTG AAA AAC AAT AAT CTG ACA GCT TGC
ATT GCT GAC TTT GGG TTG GCC TTA AAG TTC GAG GCT GGC AAG TCT GCA
GGT GAC ACC CAT GGG CAG GTT GGT ACC CGG AGG TAT ATG GCT CCA GAG
GTG TTG GAG GGT GCT ATA AAC TTC CAA AGG GAC GCA TTT CTG AGG ATA
GAT ATG TAC GCC ATG GGA TTA GTC CTA TGG GAA TTG GCT TCT CGT TGC
ACT GCT GCA GAT GGA CCC GTA GAT GAG TAC ATG TTA CCA TTT GAG GAA
GAA ATT GGC CAG CAT CCA TCT CTT GAA GAT ATG CAG GAA GTT GTT GTG
CAT AAA AAA AAG AGG CCT GTT TTA AGA GAT TAT TGG CAG AAA CAT GCA
GGA ATG GCA ATG CTC TGT GAA ACG ATA GAA GAA TGT TGG GAT CAT GAT
GCA GAA GCC AGG TTA TCA GCT GGA TGT GTA GGT GAA AGA ATT ACT CAG
ATG CAA AGA CTA ACA AAT ATC ATT ACT ACA GAG GAC ATT GTA ACA GTG
GTC ACA ATG GTG ACA AAT GTT GAC TTT CCT CCC AAA GAA TCT AGT CTA [wherein, X ′ represents a base sequence represented by ATG or hydrogen. (14) a vector containing the DNA according to (11), (15) a transformant carrying the vector according to (14), (16) A method for producing a receptor protein or a salt thereof, comprising culturing the transformant according to the above (15), producing and accumulating a receptor protein or a salt thereof in the culture, and collecting the receptor protein or a salt thereof. 17) A receptor protein or a salt thereof, which comprises culturing the transformant according to the above (15), producing and accumulating a receptor protein or a salt thereof in the culture supernatant or on the cell membrane of the transformant, and collecting the receptor protein or a salt thereof. (18) Polymerase chain reaction using a synthetic DNA containing the base sequence of the formula TACCCTCCTGTACTTGTTCCTACTCAA or TAGCCACAGGTCCACATCCACACTGGT. Method (polymerase chain rea
ction method).
A) A method for detecting the activin receptor protein according to the above (4), wherein a polymerase chain reaction method is performed using a synthetic DNA containing SEQ ID NO: 1 or SEQ ID NO: 2. And (20) an antibody against the receptor protein or a salt thereof according to the above (1). The present invention also provides (18)
(19) (i) the method according to (1), wherein the test compound is contacted with the protein or salt thereof according to (1). (1) comparing the case where the ligand is brought into contact with the protein or a salt thereof and the case (ii) where the ligand or the test compound is brought into contact with the protein or the salt thereof described in the above (1). A) a method for screening a compound or a salt thereof that inhibits or promotes the binding of the ligand to the protein or a salt thereof according to (20), which comprises the protein or a salt thereof according to the above (1) and a ligand thereto; Inhibits or promotes binding of the ligand to the protein or salt thereof described in (1) above or a known activin receptor protein or salt thereof. Compound or a screening kit for salt thereof. More specifically, (21) the protein is an amino acid sequence represented by SEQ ID NO: 5, one or more in an amino acid sequence other than SEQ ID NO: 7 in the amino acid sequence represented by SEQ ID NO: 5, (Preferably, 2 or more and 30 or less, more preferably 2 or more and 10 or less
1 or 2 or less (preferably 2 or less) in the amino acid sequence represented by SEQ ID NO: 5 in which the amino acid sequence of SEQ ID NO.
In the amino acid sequence other than SEQ ID NO: 7 in the amino acid sequence to which the amino acid sequence is added or inserted, or in the amino acid sequence represented by SEQ ID NO: 5, 1 or 2 or more (preferably 2 or more and 30 or less, more preferably 2 or more and 10
Activin receptor protein according to the above (4) and its salt, wherein the amino acid sequence of the activin receptor protein according to the above (4) contains an amino acid sequence obtained by substituting the amino acid sequence with another amino acid. When contacted with salt,
When the labeled ligand and test compound are brought into contact with the activin receptor protein or the salt thereof according to the above (4), the amount of binding to the labeled activin receptor protein or the salt thereof according to the above (4) is measured,
(23) a method for screening a compound or a salt thereof that inhibits or promotes the binding between a ligand and an activin receptor protein according to (4), (23) the activin receptor according to (4), The amount of binding of the labeled ligand to the cells when the cells were brought into contact with the cells containing the protein and when the cells were brought into contact with the cells containing the activin receptor protein described in (4) above were measured. And a method for screening a compound or a salt thereof that inhibits or promotes the binding between the ligand and the activin receptor protein according to the above (4), and (24) the labeled activin according to the above (4). When contacted with the membrane fraction of cells containing the receptor protein When the labeled ligand and the test compound are brought into contact with the membrane fraction of the cell containing the activin receptor protein described in the above (4), the amount of binding of the labeled ligand to the membrane fraction of the cell is measured, A method for screening a compound or a salt thereof that inhibits the binding between the ligand and the activin receptor protein according to the above (4); and (25) culturing the transformant according to the above (15). The activin receptor protein expressed on the cell membrane of the transformant is contacted with a labeled ligand to the activin receptor protein expressed on the cell membrane of the transformant, and the activin receptor protein expressed on the cell membrane of the transformant is cultured by culturing the transformant according to (15). Of the labeled ligand when the labeled ligand and the test compound are brought into contact with each other. A method of measuring and comparing the amount of binding to a putter protein and comparing the ligand and the activin receptor protein described in (4) above or a compound thereof which inhibits or promotes the binding, or (26) a method of screening (4) The activin receptor protein-activating compound described in (4) is brought into contact with the cells containing the activin receptor protein described in (4) above, and the activin receptor protein-activating compound and test compound described in (4) are obtained in the same manner. A ligand characterized by measuring and comparing the cell stimulating activity mediated by the activin receptor protein when brought into contact with a cell containing the activin receptor protein according to (4) and the activin receptor protein according to (4). Or salt thereof that inhibits or promotes binding to (27) a method for activating the activin receptor protein described in (4) above, wherein the compound activating the activin receptor protein described in (4) is brought into contact with the activin receptor protein expressed on the cell membrane of the transformant by culturing the transformant described in (15) And activating the activin receptor protein described in (4) and a test compound with the activin receptor protein expressed in the cell membrane of the transformant by culturing the transformant described in (15). A method for screening a compound or a salt thereof that inhibits or promotes the binding between a ligand and an activin receptor protein according to (4) above, wherein the cell stimulating activity mediated by the activin receptor protein is measured and compared. (28) The above (19) or (22) to ( 7) A compound or a salt thereof obtained by the screening method according to the above or by using the screening kit according to the above (20), or (29) a medicament comprising the compound or a salt thereof according to the above (28). (30) an activin receptor agonist or an activin receptor antagonist obtained by the screening method according to (19) or (22) to (27) or using the screening kit according to (20); A prophylactic / therapeutic agent for a neurodegenerative disease comprising the compound according to the above (28) or a salt thereof; and (32) a method for diagnosing a neurodegenerative disease using the DNA detection method according to the above (19). About.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The receptor protein of the present invention, in particular, a nervous cell-specific receptor protein is represented by SEQ ID NO:
Any amino acid sequence containing an amino acid sequence substantially the same as the amino acid sequence represented by 7 may be used, but an activin receptor protein is more preferable. The protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 7 is the protein having SEQ ID NO: 7
Means a protein having substantially the same activity as the amino acid sequence represented by Substantially the same activity includes, for example, qualitative factors such as ligand binding activity, signal transduction ability, and specificity of cell distribution. Therefore, quantitative factors such as the strength of ligand binding activity and the molecular weight of the receptor protein may be different. More specifically, for example, an amino acid sequence in which one or more (preferably 1 or 2, more preferably 1) amino acids in the amino acid sequence represented by SEQ ID NO: 7 have been deleted, : 1 or 2 in the amino acid sequence represented by 7
Or more (preferably 1 or 2, more preferably 1
Amino acids) added or inserted amino acid sequence,
Alternatively, one or more (preferably one or two, more preferably one or more) in the amino acid sequence represented by SEQ ID NO: 7
And a protein containing an amino acid sequence in which one amino acid is substituted with another amino acid. Receptor proteins include warm-blooded animals (eg, guinea pigs, rats,
Mice, rabbits, pigs, sheep, cows, monkeys, humans, etc.) (eg, stomach, pituitary, pancreas, brain,
A receptor protein derived from kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract, blood vessel, heart, etc.) or a cell;
Any protein may be used as long as it contains a protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 5, and among them, an amino acid substantially identical to the amino acid sequence represented by SEQ ID NO: 5 Those containing sequences are preferred. That is, the receptor protein of the present invention may have any length as long as it contains an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 7, and specifically, Is a protein having a length of about 8 or more amino acid sequences represented by SEQ ID NO: 7 to a protein having a length of 521 amino acid sequences represented by SEQ ID NO: 5 or more It may be of any length up to. Above all, an amino acid sequence containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 7 and having about 80 to 99.9% homology with the amino acid sequence represented by SEQ ID NO: 5 A protein having substantially the same activity as that of the protein containing the amino acid sequence represented by SEQ ID NO: 5, and particularly, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 7 Wherein the first amino acid (Met) to the 105th amino acid (Cys) of the 521 amino acid sequence represented by SEQ ID NO: 5 are N-terminal, and the 417th amino acid (Asp) to the 417th amino acid The amino acid at position 521 (Leu) is the C-terminal, which has the amino acid sequence continuously between the N-terminal and the C-terminal in the amino acid sequence represented by SEQ ID NO: 5, and The length is 417 or more The quality is preferred. Further, the first amino acid (Met) of the 521 amino acid sequence represented by SEQ ID NO: 5 may be protected or may be substituted with a hydrogen atom. Substantially the same activity includes, for example, qualitative factors such as ligand binding activity, signal transduction ability, and specificity of cell distribution. Therefore,
Quantitative factors such as strength and weakness of the ligand binding activity and the molecular weight of the receptor protein may be different.

More specifically, the receptor protein of the present invention (preferably, a neural cell-specific receptor protein,
Particularly preferably, the activin receptor protein) includes an activin receptor protein derived from mouse embryonic tumor cell P19, which contains the amino acid sequence represented by SEQ ID NO: 5. Further, as the receptor protein of the present invention, one or more (preferably 2 or more and 30 or less, in the amino acid sequence represented by SEQ ID NO: 5 other than the amino acid sequence represented by SEQ ID NO: 7) An amino acid sequence in which at least 2 and no more than 10 amino acids have been deleted, and one or more (preferably 2 or more) amino acid sequences represented by SEQ ID NO: 5 other than the amino acid sequence represented by SEQ ID NO: 7 Amino acid sequence to which 30 or less, more preferably 2 or more and 10 or less) amino acids are added or inserted,
Or one or more (preferably 2 or more and 30 or less, more preferably 2 or more and 10 or less) of the amino acid sequence represented by SEQ ID NO: 5 other than the amino acid represented by SEQ ID NO: 7 A protein containing an amino acid sequence in which an amino acid is substituted with another amino acid is also included. Further, the receptor protein of the present invention includes those in which the N-terminal Met is converted to H, and the N-terminal Met is a protecting group (for example, a C _1-6 acyl group such as a formyl group and an acetyl group (preferably a C _1-6 acyl group).
_1-6 alkanoyl group), etc.), Glu
N-terminal is cleaved in vivo and the Glu is pyroglutaminated, and the side chain of the amino acid in the molecule has an appropriate protecting group (for example, C _1-6 acyl group such as formyl group, acetyl group (preferably , A C _1-6 alkanoyl group), or a complex protein such as a so-called glycoprotein having a sugar chain bonded thereto. As the salt of the receptor protein of the present invention (preferably, a nervous cell-specific receptor protein, particularly preferably, an activin receptor protein), a physiologically acceptable acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) And tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid). Also, salts with inorganic bases (eg, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, aluminum and ammonium), organic bases (eg, trimethylamine, triethylamine, pyridine, picoline, 2,6 -Lutidine, ethanolamine,
Salts with diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N'-dibenzylethylenediamine, and the like are also used.

The receptor protein of the present invention, in particular, a nervous cell-specific receptor protein, in particular, a novel activin protein or a salt thereof can be produced from a tissue or a cell of a warm-blooded animal by a known method for purifying a protein. It can also be produced by culturing a transformant containing a DNA encoding the activin receptor protein described below. Further, it can also be produced according to the peptide synthesis method described later. SEQ ID NO: 7 of the present invention
A peptide containing an amino acid sequence substantially identical to the amino acid sequence represented by or a salt thereof can be produced according to a peptide synthesis method known per se or by cleaving the receptor protein of the present invention with an appropriate peptidase. Can be. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid that can constitute the protein of the present invention with the remaining portion, and when the product has a protecting group, removing the protecting group. Known methods for condensation and elimination of the protecting group include, for example, the methods described in (1) to (4) below. M. Bodanszky and MAOndetti, Peptide Synthesis, Interscience Publisher
s, New York (1966) Schroeder and Luebke, The Peptid
e), Academic Press, New York (1965) Nobuo Izumiya et al., Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975) Haruaki Yajima and Shunpei Sakakibara, Biochemistry Laboratory Course 1, Protein Chemistry, 205, (1977) Supervised by Haruaki Yajima, Development of Continuing Pharmaceuticals Volume 14 Peptide Synthesis Hirokawa Shoten After the reaction, the usual purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, etc. may be used as appropriate. The proteins of the present invention can be purified and isolated in combination. When the protein obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained by a salt, it can be converted to a free form by a known method. Can be.

The DNA encoding the receptor protein of the present invention, in particular, the nervous system cell-specific receptor protein, in particular, the activin receptor protein, comprises an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 7 of the present invention. Any sequence containing a nucleotide sequence encoding a receptor protein containing an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 5 may be used. It may be converted. Genome DN of warm-blooded animals (eg, humans)
A, Genomic DNA library of warm-blooded animals (eg, humans), cDN from tissues and cells of warm-blooded animals (eg, humans)
A, it may be a cDNA library derived from tissues or cells of a warm-blooded animal (eg, human) or a synthetic or semi-synthetic DNA.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. On the other hand, it can be directly amplified by RT-PCR using the mRNA fraction prepared from tissues and cells, or it can be produced by chemically synthesizing each partial nucleotide sequence and condensing them. You can also.

More specifically, the DNA encoding the activin receptor protein derived from mouse embryonic tumor cell P19 containing the amino acid sequence of SEQ ID NO: 5 includes a DNA having the base sequence represented by SEQ ID NO: 4 and the like. Is used. DNA completely encoding the receptor protein of the present invention
As a means for cloning, the DNA was amplified by PCR using a synthetic DNA primer having a partial nucleotide sequence of the receptor protein, or was incorporated into an appropriate vector.
The DNA is selected by hybridization with a DNA fragment having a part or the entire region of the human activin receptor protein or labeled with a synthetic DNA.
Hybridization methods are described, for example, in Molecular Cloning 2nd ed ,; J. Sambr
ook et.al., Cold Spring Harbor Lab. Press, (1989)
And the like. When a commercially available library is used, the procedure is performed according to the method described in the attached instruction manual. The DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or it can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. An expression vector for a receptor protein can be obtained, for example, from (a) a DNA encoding the receptor protein of the present invention using a D
It can be prepared by cutting out the NA fragment and (ii) ligating the DNA fragment downstream of the promoter in an appropriate expression vector. As vectors, plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), plasmids derived from yeast (eg, pSH19, pSH15), λ phage, etc. And animal viruses such as bacteriophage, retrovirus, vaccinia virus and baculovirus.

[0014] The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. When the host for transformation is Escherichia, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, etc., when the host is Bacillus, SPO1 promoter, SPO2 promoter,
When the host is yeast, such as the penP promoter, pHO5
Promoter, PGK promoter, GAP promoter, AD
H promoters and the like are preferred. When the host is an animal cell, an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, an SRα promoter, and the like can be used. The use of enhancers for expression is also effective. Also,
If necessary, a signal sequence suitable for the host can be added to the N-terminal side of the receptor protein. When the host is a bacterium belonging to the genus Escherichia, an alkaline phosphatase signal sequence, an OmpA signal sequence or the like is used. When the host is an animal cell, for example, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule, etc.
A signal sequence or the like can be used.

A transformant can be produced using the vector containing the DNA encoding the receptor protein constructed as described above. Examples of the host include Escherichia, Bacillus, yeast, and insects. And animal cells. Specific examples of the genus Escherichia include Escherichia coli K12 · DH1.
[Procedings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160 (1968)], J
M103 [Nucleic Acids Research, (Nucleic Ac
ids Research), 9, 309 (1981), JA221 [Journal of M.
olecular Biology)], 120, 517 (1978)], HB101
[Journal of Molecular Biology, 41
Volume, 459 (1969)], C600 [Genetics (Genetic
s), Vol. 39, 440 (1954)]. Examples of Bacillus bacteria include, for example, Bacillus
subtilis) MI114 [Gene, 24, 255 (1983)], 207
-21 [Journal of Biochemistry (Journal
of Biochemistry), 95. 87 (1984)]. As yeast, for example Saccharomyces cerevisiae ^{(Saccharomyces cerevisiae) AH22, AH22R -} , NA
87-11A, DKD-5D, 20B-12, etc. are used. As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)]. As animal cells, for example, monkey cells COS-7, Vero, Chinese hamster cells CHO, DHFR gene-deficient Chinese hamster cells CHO (dhfr ^- CHO cells), mouse L cells, mouse myeloma cells, human FL cells and the like are used. To transform Escherichia, for example, the Procesings of the National Academy of
Sciences of the USA (Proc. Natl.
Acad.Sci.USA), 69, 2110 (1972) and Gene (Gen.
e), Vol. 17, 107 (1982).

For transforming Bacillus sp., For example, Molecular & General Genetics, 168, 111 (197)
This is performed according to the method described in 9). To transform yeast, for example, the Procesings of the National Academy of Sciences of Science
The USA (Proc. Natl. Acad. Sci. USA) 75, 19
29 (1978). Transformation of insect cells is performed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988). To transform animal cells, for example, Virology, 52, 456 (1
973). Thus, a transformant transformed with the expression vector containing the DNA encoding the activin receptor protein is obtained.

When culturing a transformant whose host is a genus Escherichia or Bacillus, a liquid medium is suitable as a medium to be used for the cultivation. A carbon source, a nitrogen source, an inorganic substance and the like are contained. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, 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, an M9 medium containing glucose and casamino acid [Miller, Journal
Journal of Experiments in MolecularGetics
netics), 431-433, Cold Spring Harbor Laboratory,
New York 1972] is preferred. If necessary, an agent such as 3β-indolylacrylic acid can be added to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, culturing is usually performed at about 15 to 43 ° C.
The reaction is performed for 3 to 24 hours, and if necessary, ventilation or stirring can be added. When the host is Bacillus, the culture is usually about 30
The reaction is carried out at 4040 ° C. for about 6 to 24 hours, and if necessary, aeration and stirring can be added. When culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Bostian, K. et al. L. Processings of the National Academy of Sciences of the USA (Proc.Nat
I. Acad. Sci. USA), Volume 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, G. et al. A. Processings of the National Academy of Sciences of the USA (Proc. Natl. Ac
ad.Sci.USA), 81, 5330 (1984)]. Preferably, the pH of the medium is adjusted to about 5-8. Culture is usually about
Perform at about 20-35 ° C for about 24-72 hours, adding aeration and stirring as needed. When culturing a transformant whose host is an insect, the medium used is Grace's Insect Medium (Grace, T .;
CC, Nature, 195, 788 (1962)) to which an additive such as immobilized 10% bovine serum is appropriately added. Preferably, the pH of the medium is adjusted to about 6.2-6.4. The cultivation is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and / or agitation are added as necessary. When culturing a transformant in which the host is an animal cell, the medium may be, for example, about 5 to 20.
% MEM medium containing fetal bovine serum [Seienc
e), Vol. 122, 501 (1952)], DMEM medium [Virology, Vol. 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association (The Jounal of the American Medical)
Association) Volume 199. 519 (1967)], 199 medium [Proceeding of the Societe for the Biological Medicine (Proceeding of the Society)
y for the Biological Medicine), 73, 1 (1950)]
Are used. Preferably, the pH is between about 6 and 8. The cultivation is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and aeration and stirring are added as necessary. Separation and purification of the receptor protein from the above culture can be performed, for example, by the following method.

When the receptor protein of the present invention is extracted from cultured cells or cells, the cells or cells are collected by a known method after culture, suspended in an appropriate buffer, and then subjected to ultrasonication, lysozyme and / or lysozyme. Alternatively, a method of disrupting cells or cells by freeze-thawing or the like and then obtaining a crude extract of the receptor protein by centrifugation or filtration may be used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (trade name). When the receptor protein is secreted into the culture solution, after the culture is completed, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected. Purification of the receptor protein contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods. These known separation and purification methods include methods using solubility such as salting-out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, mainly molecular weight. Method using difference in charge, method using charge difference such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and use of difference in hydrophobicity such as reverse phase high performance liquid chromatography A method utilizing a difference between isoelectric points, such as an isoelectric focusing method, is used.

When the thus obtained receptor protein is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when the receptor protein is obtained in a salt form, it is a known method. Alternatively, it can be converted to a free form or another salt by a method analogous thereto. The receptor protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein-modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used. The activity of the receptor protein thus produced can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.

[0020] DN encoding the receptor protein of the present invention
A and the receptor protein, a method for determining a ligand for the receptor protein of the present invention, obtaining antibodies and antisera, constructing a recombinant receptor protein expression system,
Development of a receptor binding assay system using the same expression system and screening of candidate drug compounds, drug design based on comparison with structurally similar ligands / receptors, probes for genetic diagnosis, PCR
It can be used as a reagent in the preparation of primers and the like, and can also be used as a drug for gene therapy and the like. In particular, a receptor agonist or antagonist specific to a warm-blooded animal such as a human can be screened by a receptor binding assay system using the recombinant receptor protein expression system of the present invention, and the agonist or antagonist can be used for various diseases. It can be used as a prophylactic / therapeutic agent.

The receptor protein of the present invention, in particular, a nervous cell-specific receptor protein, especially an activin receptor protein or a salt thereof, a receptor protein, particularly a nervous cell-specific receptor protein, especially a DNA encoding an activin receptor protein and a receptor protein The use of the antibody against is described more specifically below. (1) Method for Determining Ligand for Receptor Protein of the Present Invention The receptor protein of the present invention or a salt thereof is useful as a reagent for searching for or determining a ligand for the receptor protein of the present invention. That is, the present invention provides a method for determining a ligand for the receptor protein of the present invention, which comprises contacting the receptor protein of the present invention or a salt thereof with a test compound. Examples of the test compound include known ligands (eg, activin, inhibin, TGFβ, OP-1 gene product, Vg-1 gene product) and, for example, warm-blooded animals (eg, mouse, rat, pig, cow, sheep, (Monkey, human, etc.), cell culture supernatant, and the like. For example, the tissue extract, cell culture supernatant, or the like is added to the receptor protein of the present invention, and fractionation is performed while measuring cell stimulating activity and the like, so that a single ligand can be finally obtained. Specifically, the ligand determination method of the present invention uses the receptor protein of the present invention or a salt thereof, or constructs a recombinant receptor protein expression system and uses a receptor binding assay system using the expression system. Thus, a compound having a cell stimulating activity (eg, an activity of promoting or suppressing growth of a cell, phosphorylation of an intracellular protein, etc.) by binding to a receptor protein (eg, a peptide, a protein, a non-peptide compound, a synthetic compound) , Fermentation products, etc.) or its salts. In the ligand determination method of the present invention, when the receptor protein of the present invention or a salt thereof is contacted with a test compound, for example, the amount of a test compound bound to the receptor protein or a salt thereof,
It is characterized by measuring cell stimulating activity and the like.

More specifically, the present invention measures the binding amount of a labeled test compound to a protein or a salt thereof when the test compound is contacted with the receptor protein of the present invention or a salt thereof. A method for determining a ligand for a receptor protein, comprising: contacting a labeled test compound with a cell containing the receptor protein of the present invention or a membrane fraction of the cell; A method for determining a ligand for a receptor protein, which comprises measuring the amount of binding to the membrane fraction; and culturing a transformant containing a DNA encoding the receptor protein of the present invention with a labeled test compound. The receptor for a labeled test compound when contacted with the receptor protein expressed above. A method for determining a ligand for a receptor protein, which comprises measuring the amount of binding to a receptor protein, and a cell stimulating activity via a receptor protein when a test compound is brought into contact with a cell containing the receptor protein of the present invention ( For example, a method for determining a ligand for a receptor protein, which comprises measuring the activity of promoting or suppressing growth of a protein, phosphorylation of an intracellular protein, and the like, and a test compound comprising a DNA encoding the receptor protein of the present invention. A cell stimulating activity via the receptor protein (for example, promoting or suppressing growth promotion, phosphorylation of intracellular protein, etc.) when the transformant containing the protein is brought into contact with a receptor protein expressed on the cell membrane by culturing. Activity, etc.) -A method for determining a ligand for a protein is provided.

A specific description of the ligand determination method of the present invention will be given below. First, as the receptor protein used in the ligand determination method, any receptor protein of the present invention, that is, any protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 7 is used. Alternatively, a receptor protein expressed in large amounts using animal cells is suitable. The above-mentioned method is used for producing a receptor protein, but it can be carried out by expressing a DNA encoding the protein in mammalian cells or insect cells. CDNA is used as the DNA fragment encoding the target portion, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. To introduce a DNA fragment encoding a receptor protein into host animal cells and express them efficiently,
The DNA fragment is used as a nuclear polyhedrosis virus belonging to baculovirus using an insect as a host.
It is preferable to incorporate the promoter into the downstream of the polyhedrin promoter of NPV), the promoter derived from SV40, the retrovirus promoter, the metallothionein promoter, the human heat shock promoter, the cytomegalovirus promoter, and the SRα promoter. The amount and quality of the expressed receptor can be examined by a method known per se. For example, in the literature [Nambi, P. et al., The Journal of Biological Chemistry (Journal of
Biological Chemistry), 267, 19555-19559, (1
992)]. Therefore, in the ligand determination method of the present invention, the receptor protein or a partial peptide of the receptor protein may be a receptor protein or a partial peptide of the receptor protein purified according to a method known per se, A cell containing the protein may be used, or a membrane fraction of the cell containing the protein may be used. When cells containing a receptor protein are used in the ligand determination method of the present invention, the cells may be immobilized with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a method known per se.

Cells containing a receptor protein, especially a nervous system cell-specific receptor protein, especially an activin receptor protein, refer to host cells that have expressed the receptor protein. Examples of such host cells include Escherichia coli, Bacillus subtilis, yeast, and the like. Examples include insect cells and animal cells. The membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. Cell crushing methods include crushing cells with a Potter-Elvehjem homogenizer, crushing with a Waring blender or Polytron (Kinematica), crushing with ultrasonic waves, and blowing cells from a thin nozzle while applying pressure with a French press. Crushing and the like.
For fractionation of cell membranes, fractionation by centrifugal force, such as fractionation centrifugation and density gradient centrifugation, is mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (generally about 1 to 10 minutes), and the supernatant is further centrifuged at a high speed (150 rpm to 150 rpm).
(00 rpm to 30,000 rpm) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in the expressed receptor protein and membrane components such as cell-derived phospholipids and membrane proteins. The amount of the receptor protein in the cells or membrane fraction containing the receptor protein is 10 ² per cell.
It is preferably from 10 to 10 ⁸ molecules, and more preferably from 10 ⁵ to 10 ⁷ molecules. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. .

In order to carry out the above-mentioned methods for determining a ligand which binds to a receptor protein, an appropriate receptor fraction and a labeled test compound are required.
The receptor fraction is preferably a natural activin receptor fraction or a recombinant receptor fraction having an activity equivalent thereto. Here, the equivalent activity indicates an equivalent ligand binding activity and the like. The labeled test compound, ^[3 H], ^[125 I], ^[14 C]
Activin, inhibin, TG labeled with [ ¹³⁵ S]
Fβ, OP-1 gene product, Vg-1 gene product and the like are preferred.

Specifically, to determine a ligand that binds to a receptor protein, a cell or a membrane fraction of the cell containing the receptor protein is first suspended in a buffer suitable for the determination method. Prepare a standard. The buffer may have a pH of 4 to 10 (preferably a pH of 6 to
Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor, such as the phosphate buffer and Tris-HCl buffer of 8). In addition, in order to reduce non-specific binding, CHAPS, Tween-80 (trade name) (Kao-Atlas), digitonin, surfactants such as deoxycholate, and various proteins such as bovine serum albumin and gelatin are used as buffers. Can be added. Furthermore, to reduce the degradation of receptors and ligands by proteases
Protease inhibitors such as PMSF (phenylmethanesulfonyl fluoride), leupeptin, E-64 (manufactured by Peptide Research Laboratories), and pepstatin can also be added. 0.01ml
A fixed amount (5000 cpm to 50,000) is added to ~ 10 ml of the receptor solution.
0 cpm) of a test compound labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C] or the like. A reaction tube containing a large excess of unlabeled test compound is also prepared to determine the amount of non-specific binding (NSB). The reaction is carried out from 0 ° C to 50 ° C, preferably from 4 ° C
The reaction is carried out at 37 ° C. for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction, the mixture is filtered through a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. Non-specific binding (N
A test compound whose count (B-NSB) minus SB) exceeds 0 cpm can be selected as a ligand for the receptor protein of the present invention.

In order to carry out the above methods (1) and (2) for determining a ligand that binds to a receptor protein, a cell stimulating activity (for example, growth promotion,
Activity such as promoting or inhibiting intracellular protein phosphorylation) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured in a multiwell plate or the like. Prior to ligand determination, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant to generate The quantified product is quantified according to each method.
When the production of a substance (for example, plasminogen activator inhibitor 1, fibronectin, etc.) as an indicator of the cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an inhibitor for the degrading enzyme is added to the assay. May be performed. The kit for determining a ligand that binds to the receptor protein of the present invention contains the receptor protein of the present invention or a salt thereof, a cell containing the receptor protein of the present invention, or a membrane fraction of a cell containing the receptor protein of the present invention. Things.

Examples of the kit for determining a ligand of the present invention include the following. 1． Ligand determination reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
5% bovine serum albumin (Sigma). The solution may be sterilized by filtration through a 0.45 μm filter and stored at 4 ° C., or may be prepared at use. CHO cells expressing the receptor protein were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well and incubated at 37 ° C., 5% CO ₂ and 95% air.
Cultured for days. Labeled test compound A commercially available compound labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ¹³⁵ S] or the like, or an aqueous solution labeled by an appropriate method, is used at 4 ° C. or −20 ° C. Store at ℃ and dilute to 1 μM with a measurement buffer before use. For test compounds that are poorly soluble in water, dimethylformamide, DMSO,
Dissolve in methanol and the like. Labeled test compound The same labeled compound is prepared at a concentration 100 to 1000 times higher. 2． Assay Method CHO cells expressing a receptor protein cultured on a 12-well tissue culture plate are washed twice with 1 ml of a measuring solution, and 490 μl of a measurement buffer is added to each well. 5 μl of the labeled test compound is added and reacted at room temperature for 1 hour. To determine the amount of non-specific binding, use an unlabeled test compound.
Add 5 μl. Remove the reaction solution and wash three times with 1 ml of wash buffer.
The labeled test compound bound to the cells is dissolved in 0.2N NaOH-1% SDS, and mixed with 4 ml of liquid scintillator A (Wako Pure Chemical Industries, Ltd.). Liquid scintillation counter (Beckman)
The radioactivity is measured using.

(2) Agent for Prevention / Treatment of Receptor Protein Deficiency of the Present Invention In the above method (1), if a ligand for the receptor protein of the present invention is identified, the method of the present invention may be performed according to the action of the ligand. The DNA encoding the receptor protein can be used as a preventive or therapeutic agent for activin receptor protein deficiency. For example, when there is a patient in whom the physiological action of the ligand cannot be expected due to a decrease in the receptor protein of the present invention in vivo, (a) administering the DNA encoding the receptor protein of the present invention to the patient and expressing the same. By or (b)
DN encoding the receptor protein of the present invention in brain cells, etc.
After the insertion and expression of A, the amount of the receptor protein in the brain cells of the patient can be increased, for example, by transplanting the brain cells into the patient, so that the effect of the ligand can be sufficiently exerted. Therefore, the DNA encoding the receptor protein of the present invention can be used as a safe and low-toxic agent for preventing or treating the receptor protein deficiency of the present invention. When the DNA of the present invention is used as the above-mentioned therapeutic agent, the DNA may be used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like, followed by a routine procedure. it can. For example, tablets, capsules, elixirs, microcapsules, and the like, optionally coated with sugar, aseptic, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections. For example, the DNA of the present invention is mixed with a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, binder, etc. in a unit dosage form generally required for the practice of the formulation. Can be manufactured by The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.

Examples of additives which can be mixed with tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. And a lubricating agent such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, reddish oil or cherry, and the like. When the preparation unit form is a capsule, a liquid carrier such as oil and fat can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice of dissolving or suspending the active substance in vehicles, such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil and the like. Aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like, and suitable solubilizing agents, for example, Alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), non-ionic surfactants (eg, polysorbate)
80 (trade name), HCO-50), etc. The oily liquid includes sesame oil, soybean oil and the like, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent. Also, a buffer (for example, phosphate buffer,
Sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants And the like. The prepared injection is usually filled in a suitable ampoule. Since the thus obtained preparation is safe and has low toxicity, it can be administered, for example, to warm-blooded mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, humans, etc.). it can. The dosage of the DNA varies depending on the symptoms and the like, but in the case of oral administration, generally in adults (as 60 kg), about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 50 mg per day. It is about 1.0-20 mg.
In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc. About 0.01 to 30 mg, preferably about 0.
It is convenient to administer about 1 to 20 mg, more preferably about 0.1 to 10 mg, by intravenous injection. For other animals,
The amount converted per 60 kg can be administered.

(3) Method for Quantifying Ligands for the Receptor Protein of the Present Invention Since the receptor protein of the present invention or a salt thereof has a binding property to a ligand, the ligand concentration in a living body can be quantified with high sensitivity. Can be. The quantification method of the present invention can be used, for example, by combining it with a competition method. That is, the ligand concentration in the test sample can be measured by bringing the test sample into contact with the receptor protein of the present invention or a salt thereof. In particular,
For example, it can be used according to the following method or a method analogous thereto. Hiroshi Irie "Radio immunoassay" (Kodansha, Showa 49
Published by Hiroshi Irie, "Continuing Radio Immunoassay" (Kodansha, Showa)
(4) Screening method for compounds that inhibit or promote the binding of the receptor protein of the present invention to a ligand. Using the receptor protein of the present invention or a salt thereof, or constructing a recombinant receptor protein expression system, Compounds (eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof that inhibit or promote the binding of ligand to receptor protein by using a receptor binding assay system using the expression system Can be screened. Such compounds include a compound having a cell stimulating activity (eg, an activity of promoting or suppressing proliferation, phosphorylation of an intracellular protein, etc.) via a receptor (a so-called receptor agonist of the present invention) and a cell stimulating activity. And compounds having no activity (so-called receptor antagonists of the present invention). That is, the present invention relates to (i) a case where a ligand is brought into contact with the receptor protein of the present invention or a salt thereof, and (ii) a case where a ligand and a test compound are brought into contact with the receptor protein or a salt thereof of the present invention. A method for screening a compound or a salt thereof that inhibits or promotes binding between a ligand and a receptor protein of the present invention, characterized by performing a comparison. In the screening method of the present invention, (i) the case where a ligand is brought into contact with the receptor protein of the present invention or a salt thereof, and (ii) the case where the receptor protein or a salt thereof of the present invention is used.
When contacting the ligand and the test compound,
For example, the amount of ligand binding to the receptor protein or a salt thereof, the cell stimulating activity, and the like are measured and compared.

More specifically, the present invention relates to a method of contacting a labeled ligand with the receptor protein of the present invention or a salt thereof, and a method of contacting a labeled ligand and a test compound with the receptor protein of the present invention or a salt thereof. A method for screening a compound or a salt thereof that inhibits or promotes the binding between a ligand and a receptor protein of the present invention, wherein the amount of binding of the labeled ligand to the protein or a salt thereof is measured and compared. Contacting a labeled ligand with a cell containing the receptor protein of the present invention or a membrane fraction of the cell;
When the labeled ligand and the test compound are brought into contact with the cell containing the receptor protein of the present invention or the membrane fraction of the cell, the amount of the labeled ligand bound to the cell or the membrane fraction is measured and compared. A method for screening a compound or a salt thereof that inhibits or promotes binding of a ligand to a receptor protein of the present invention, and culturing a transformant containing a DNA encoding the receptor protein of the present invention with a labeled ligand. And a receptor expressed on a cell membrane by culturing a transformant containing a DNA encoding the receptor protein of the present invention with a labeled ligand and a test compound. Receptor of labeled ligand when contacted with protein A method for screening a compound or a salt thereof that inhibits or promotes the binding between a ligand and a receptor protein of the present invention, which comprises measuring and comparing the amount of binding to a protein, and a compound that activates the receptor protein of the present invention (for example, , A ligand for the receptor protein of the present invention, etc.)
Was contacted with cells containing the receptor protein of the present invention, and when the compound that activates the receptor of the present invention and a test compound were contacted with cells containing the receptor protein of the present invention, A cell stimulating activity (eg, an activity of promoting or suppressing proliferation, phosphorylation of an intracellular protein, etc.) is measured and compared, and the binding between a ligand and a receptor protein of the present invention is inhibited or promoted. A method for screening a compound or a salt thereof, and culturing a transformant containing a DNA encoding the receptor protein of the present invention with a compound that activates the receptor of the present invention (eg, a ligand for the receptor protein of the present invention). Of the present invention and the receptor of the present invention. Receptor-mediated cell stimulating activity when a compound that activates a sceptor and a test compound are brought into contact with a receptor protein expressed on a cell membrane by culturing a transformant containing a DNA encoding the receptor protein of the present invention. (Eg, an activity of promoting or suppressing growth promotion, phosphorylation of an intracellular protein, etc.) and comparing the ligand with a compound that inhibits or promotes the binding between the ligand and the receptor protein of the present invention, or a compound thereof. A method for screening a salt is provided.

The specific description of the screening method of the present invention is as follows. First, as the receptor protein used in the screening method of the present invention, any receptor protein of the present invention, in particular, a nervous cell-specific receptor protein, particularly an activin receptor protein or a salt thereof, is used. Alternatively, a membrane fraction of an organ of a warm-blooded animal is preferred. However, since human organs are particularly difficult to obtain, a receptor protein or a salt thereof which is expressed in a large amount using a recombinant is suitable for screening.
The above-mentioned method is used for producing a receptor protein. For example, it can be carried out by expressing a DNA encoding the protein in mammalian cells or insect cells. Complementary DNA is used as the DNA fragment encoding the target portion, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. In order to introduce a DNA fragment encoding a receptor protein into host animal cells and express them efficiently, the DNA fragment is required to be a nuclear polyhedrosis virus (NPV) belonging to a baculovirus using an insect as a host. It is preferable to incorporate the promoter into the downstream of the polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human / human shock promoter, cytomegalovirus promoter, SRα promoter and the like. The amount and quality of the expressed receptor can be examined by a method known per se. For example, the literature [Nambi, P. et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19]
559, 1992].

Accordingly, in the screening method of the present invention, the receptor protein or a salt thereof may be a receptor protein or a salt thereof purified according to a method known per se or a cell containing the protein. May be used, or a membrane fraction of cells containing the protein may be used. When cells containing a receptor protein are used in the screening method of the present invention, the cells may be immobilized with glutaraldehyde, formalin, or the like. The immobilization method can be performed according to a method known per se. The cell containing the receptor protein refers to a host cell that has expressed the receptor protein. Examples of the host cell include Escherichia coli, Bacillus subtilis, yeast, insect cells, and animal cells. The membrane fraction refers to a fraction abundant in cell membrane obtained by disrupting cells and then obtained by a method known per se. Cell crushing methods include crushing cells with a Potter-Elvehjem homogenizer, crushing with a Waring blender or Polytron (Kinellmatica), sonication,
Examples include crushing by ejecting cells from a fine nozzle while applying pressure by a French press or the like. For fractionation of cell membranes, fractionation by centrifugal force, such as fractionation centrifugation and density gradient centrifugation, is mainly used. For example,
The cell lysate is centrifuged at a low speed (500 rpm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further centrifuged (15000 rp).
(m-30000 rpm) for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in the expressed receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.

The amount of the receptor protein in the cell or membrane fraction containing the receptor protein is 10 ² to 10 ⁸ per cell.
It is preferably a molecule, more preferably 10 ⁵ to 10 ⁷ molecules. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to measure a large number of samples in the same lot. . In order to screen for a compound that inhibits the binding between the ligand and the receptor of the present invention, a suitable receptor fraction and a labeled ligand are required to carry out the above-mentioned steps. The receptor fraction is preferably a natural receptor fraction or a recombinant receptor fraction having an activity equivalent thereto. Here, the equivalent activity indicates an equivalent ligand binding activity and the like. As the labeled ligand, a labeled ligand, a labeled ligand analog compound, or the like is used. For example ^[3 H], ^[125 I] ^[14 C], ^[135 S]
Such a labeled ligand can be used.

Specifically, to screen for a compound that inhibits the binding between a ligand and a receptor protein, first, cells containing the receptor protein or a membrane fraction of the cells are suspended in a buffer suitable for screening. To prepare a receptor preparation. The buffer may be any buffer such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-HCl buffer, which does not inhibit the binding between the ligand and the receptor. In addition, CHAP was used to reduce non-specific binding.
Surfactants such as S, Tween-80 (trade name) (Kao-Atlas Co., Ltd.), digitonin, and deoxycholate can also be added to the buffer. Furthermore, a protease inhibitor such as PMSF, leupeptin, E-64 (manufactured by Peptide Research Institute), or pepstatin can be added for the purpose of suppressing the degradation of the receptor or ligand by the protease. 0.01
Add a fixed amount (5000 cpm-5
00000 cpm) of the labeled ligand is added, and at the same time, 10 ⁻⁴ M to ^{10 −10} M of the test compound is allowed to coexist. A reaction tube containing a large excess of unlabeled ligand is also prepared to determine the amount of non-specific binding (NSB). Reaction from 0 ° C to 5
The reaction is carried out at 0 ° C., preferably 4 ° C. to 37 ° C., for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction, the mixture is filtered through a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. Count from non-antagonistic substance (B0) to non-specific binding (NSB)
Assuming that the count (B0-NSB) obtained by subtracting is 100%, a test compound having a specific binding amount (B-NSB) of, for example, 50% or less can be used as a candidate substance having an antagonistic inhibitory ability or a specific binding amount ( A test compound having a B-NSB) of, for example, 150% or more can be selected as a candidate compound having a binding promoting ability.

In order to carry out the above-mentioned methods (1) and (2) for screening a compound that inhibits or promotes the binding of a ligand to the receptor protein of the present invention, a cell stimulating activity (for example, growth promotion, intracellular protein Activity that promotes or inhibits phosphorylation, etc.) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured in a multiwell plate or the like. When performing screening, the cells were exchanged with a fresh medium or an appropriate buffer that does not show toxicity for cells in advance, and test compounds were added and incubated for a certain period of time. The product is quantified according to the respective method.
When the production of a substance (for example, plasminogen activator inhibitor 1, fibronectin, etc.) as an indicator of the cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an inhibitor for the degrading enzyme is added to the assay. May be performed. In order to perform screening by measuring cell stimulating activity, cells expressing an appropriate receptor protein are required. Examples of cells expressing the receptor protein of the present invention include a cell line having a natural receptor protein of the present invention (eg, mouse embryonic tumor cell P19), a recombinant receptor protein expressing cell line, and the like. Is desirable. Test compounds include, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like, and these compounds may be novel compounds. Alternatively, a known compound may be used.

A screening kit for a compound or a salt thereof that inhibits or promotes the binding of a ligand to the receptor protein of the present invention includes a receptor protein or a salt thereof, a partial peptide or a salt thereof, a receptor protein of the present invention. Or the membrane fraction of cells containing the receptor protein of the present invention.

Examples of the screening kit of the present invention include the following. 1． Screening reagent Measurement buffer and washing buffer Hanks' Balanced Salt Solution (manufactured by Gibco)
5% bovine serum albumin (Sigma). The solution may be sterilized by filtration through a 0.45 μm filter and stored at 4 ° C., or may be prepared at use. Receptor standard CHO cells expressing the receptor protein were subcultured on a 12-well plate at 5 × 10 ⁵ cells / well, and incubated at 37 ° C., 5% CO ₂ and 95% air.
Cultured for days. Labeled ligand A commercially available solution of a ligand labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc. at 4 ° C or -20 ° C
And dilute to 1 μM with a measuring buffer before use. Ligand standard solution Ligand is dissolved at 1 mM in PBS containing 0.1% bovine serum albumin (Sigma) and stored at -20 ° C.

2. Assay Method CHO cells expressing a receptor protein cultured on a 12-well tissue culture plate are washed twice with 1 ml of assay buffer, and 490 μl of assay buffer is added to each well. After adding 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M, add 5 μl of a labeled ligand, and react at room temperature for 1 hour. To determine the amount of non-specific binding, use 10 ^-3 M instead of test compound
Add 5 μl of the ligand. Remove the reaction solution and wash three times with 1 ml of wash buffer.
The labeled ligand bound to the cells is dissolved in 0.2N, NaOH-1% SDS, and mixed with 4 ml of liquid scintillator A (Wako Pure Chemical Industries, Ltd.). Liquid scintillation counter (Beckman)
Radioactivity was measured using Percent Maximum Binding
(PMB) is obtained by the following equation [Equation 1].

## EQU00001 ## PMB: [(B-NSB) / (BO-NSB)]. Times.100 PMB: Percent Maximum Binding B: Value when a sample is added NSB: Non-specific Binding (non-specific binding amount) B0: Maximum binding amount

A compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound that inhibits or promotes the binding of a ligand to the receptor of the present invention, in particular, a nervous cell-specific receptor, especially an activin receptor. Specifically, a compound having a cell stimulating activity via a receptor or a salt thereof (so-called receptor agonist) or a compound having no stimulating activity (so-called receptor antagonist). Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like. These compounds may be novel compounds or known compounds. Since the receptor agonist has the same activity as the physiological activity of the ligand for the receptor protein of the present invention, it is safe and low toxic according to the ligand activity, especially a drug for preventing and treating neurodegenerative diseases. Useful as Conversely, since a receptor antagonist can suppress the physiological activity of a ligand for the receptor protein of the present invention, it can be used as a safe and low-toxicity pharmaceutical composition for inhibiting the ligand activity, particularly as a preventive or therapeutic drug for neurodegenerative diseases. Useful.

When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional method. For example, tablets or capsules coated with sugar, capsules, elixirs, microcapsules, or the like, or sterile solution or suspension with water or other pharmaceutically acceptable liquids, if necessary. It can be used parenterally in the form of injections such as liquids. For example, the compound or a salt thereof is mixed with physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders and the like in a unit dosage form required for generally accepted pharmaceutical practice. Can be manufactured. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained. tablet,
Examples of additives that can be mixed with capsules include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, alginic acid, and the like. And lubricating agents such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, reddish oil or cherry. When the preparation unit form is a capsule, a liquid carrier such as oils and fats can be further contained in the above-mentioned type of material. Sterile compositions for injection can be formulated according to normal pharmaceutical practice of dissolving or suspending the active substance in vehicles, such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil and the like. Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and suitable solubilizing agents. For example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 (TM),
HCO-50) may be used in combination. The oily liquid includes sesame oil, soybean oil and the like, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as a solubilizing agent.
In addition, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage Agents (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection is usually filled in a suitable ampoule. Since the thus obtained preparation is safe and has low toxicity, it can be administered, for example, to warm-blooded mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, humans, etc.). it can.

The dose of the compound or a salt thereof varies depending on the symptoms and the like.
00 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 50 mg
20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom administration method, and the like. About 0.01 to 30 mg per
Preferably about 0.1-20 mg, more preferably about 0.1-10
Conveniently, about mg is administered by intravenous injection. In the case of animals other than humans, an amount equivalent to the equivalent amount per 60 kg can be administered.

(5) Production of Antibodies or Antisera Against the Receptor Protein of the Present Invention, Partial Peptides or Their Salts Antibodies to the Receptor Protein of the Present Invention or Their Salts (eg, Polyclonal Antibodies, Monoclonal Antibodies)
Alternatively, an antiserum can be produced by using the receptor protein of the present invention or a salt thereof as an antigen according to a method for producing an antibody or an antiserum known per se. For example, a monoclonal antibody can be produced according to the method described below. [Preparation of Monoclonal Antibody] (a) Preparation of Monoclonal Antibody-Producing Cell The receptor protein of the present invention, a partial peptide thereof, or a salt thereof (hereinafter, sometimes abbreviated as receptor)
Is administered to a warm-blooded animal by itself or together with a carrier or diluent at a site where antibody production is possible by administration.
Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of the warm-blooded animal to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and mice and rats are preferably used. When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with the antigen, for example, an individual with an antibody titer recognized from a mouse is selected, and spleen or lymph nodes are collected 2 to 5 days after the final immunization and contained therein. By fusing the antibody-producing cells obtained with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting a labeled receptor described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation is performed by a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (19)
75)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, and the like, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is
The ratio is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG
6000) is added at a concentration of about 10 to 80%, and incubation at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes allows efficient cell fusion.

Various methods can be used for screening anti-receptor antibody-producing hybridomas. For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a receptor antigen is adsorbed directly or together with a carrier, and then, Add anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody is used if cells used for cell fusion are mice) or protein A, and detect anti-receptor monoclonal antibody bound to solid phase A method of adding a hybridoma culture supernatant to a solid phase on which an anti-immunoglobulin antibody or protein A is adsorbed, adding a receptor labeled with a radioactive substance, an enzyme, or the like, and detecting an anti-receptor monoclonal antibody bound to the solid phase And so on. Selection of the anti-receptor monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Normal
It is performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as a hybridoma can grow. For example, 1-20%
RPMI 1640 medium, preferably containing 10-20% fetal calf serum,
A GIT medium containing 1 to 10% fetal bovine serum (manufactured by Wako Pure Chemical Industries, Ltd.) or a serum-free medium for hybridoma culture (SFM-101, manufactured by Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is
Usually it is 20 to 40 ° C, preferably about 37 ° C. The culture time is
Usually 5 days to 3 weeks, preferably 1 week to 2 weeks. The cultivation is usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the anti-receptor antibody titer in the antiserum described above.

(B) Purification of Monoclonal Antibody Separation and purification of the anti-receptor monoclonal antibody can be carried out in the same manner as in the separation and purification of normal polyclonal antibodies. Method, electrophoresis, adsorption / desorption using an ion exchanger (eg, DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase, or active antibody such as protein A or protein G to collect only antibodies and bind And a specific purification method for obtaining an antibody). (A) and (b) above
The receptor antibody of the present invention produced according to the method of
Since the receptor can be specifically recognized, it can be used for quantification of a receptor in a test solution, particularly for quantification by a sandwich immunoassay.

That is, the present invention provides, for example, (i) competitively reacting an antibody reacting with the receptor of the present invention with a test solution and a labeled receptor, and determining the ratio of the labeled receptor bound to the antibody. A method for quantifying a receptor in a test solution, wherein (ii) reacting the test solution with an antibody insolubilized on a carrier and a labeled antibody simultaneously or successively, In the method for quantifying a receptor in a test solution, the activity of the above labeling agent is measured, wherein one antibody is an antibody that recognizes the N-terminal or C-terminal of the receptor, and the other antibody is SEQ ID NO: : 7
The present invention provides a method for quantifying a receptor in a test liquid, which is an antibody that reacts with the amino acid sequence of The receptor can be measured using a monoclonal antibody that recognizes the receptor of the present invention (hereinafter, may be referred to as an anti-receptor antibody), and can also be detected by tissue staining or the like. For these purposes, the antibody molecule itself may be used, or F (ab ') ² , Fab' or Fab fraction of the antibody molecule may be used. The measuring method using the antibody of the present invention is not particularly limited, and the amount of the antibody, the antigen, or the antibody-antigen complex corresponding to the amount of the antigen (for example, the amount of the receptor) in the liquid to be measured can be determined chemically or physically. Any measurement method may be used as long as it is detected by standard means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry,
A competitive method, an immunometric method and a sandwich method are preferably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity. Examples of the labeling agent used in the measurement method using a labeling substance include a radioisotope, an enzyme, a fluorescent substance, and a luminescent substance. As radioisotopes, for example, ( ¹²⁵ I), ( ¹³¹ I),
^[3 H], ^[14 C] and the like, examples of the enzyme, large preferably stable and specific activity, for example beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like, Examples of the fluorescent substance include fluorescamine and fluorescein isothiocyanate.
Luminol derivatives, luciferin, lucigenin, and the like, respectively. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass. In the sandwich method, the test solution is reacted with the insolubilized anti-receptor antibody (primary reaction), and further reacted with the labeled anti-receptor antibody (secondary reaction), and then the activity of the labeling agent on the insolubilized carrier is measured. This makes it possible to quantify the amount of receptor in the test solution. The primary and secondary reactions can be performed in reverse order,
It may be performed simultaneously or at a different time. The labeling agent and the method of insolubilization can be in accordance with those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity and the like. You may. In the method for measuring a receptor by the sandwich method of the present invention, an anti-receptor antibody used for the primary reaction and the secondary reaction is preferably an antibody having a different binding site to the receptor. That is, the antibody used in the primary reaction and the secondary reaction, for example, when the antibody used in the secondary reaction recognizes the C-terminal or N-terminal of the receptor,
As the antibody used in the primary reaction, an antibody that recognizes the amino acid sequence represented by SEQ ID NO: 7 is preferably used.

The receptor antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, a nephrometry and the like. In the competitive method, the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody. (B / F
Separation), the amount of labeling in either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, or a solid-phased antibody is used as the first antibody, or A solid-phase method using a soluble first antibody and a solid-phased antibody as the second antibody is used. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated. Is reacted with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
In nephelometry, the amount of insoluble precipitates generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying these individual immunological measurement methods to the measurement method of the present invention, no special conditions, operations and the like need to be set. Receptor measurement systems may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions operating methods in each method. For details of these general technical means, it is possible to refer to reviews, books, and the like [for example, Hiroe Irie, "Radio Immunoassay"]
(Kodansha, published in 1974), edited by Hiro Irie, "Radio Immunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al., "Enzyme immunoassay" (Medical Publishing, published in 1978), edited by Eiji Ishikawa, "Enzyme immunoassay" (2nd edition) (Medical Publishing, published in 1982), Eiji Ishikawa et al. "Enzyme immunoassay" (3rd edition)
Edition) (Medical Shoin, published in 1987) "Methods in ENZYMOL
OGY ”Vol.70 (Immunochemical Techniques (Part
A)), Ibid. Vol. 73 (Immunochemical Techniques (Part
B)), Ibid., Vol. 74 (Immunochemical Techniques (Par
t C)), ibid. Vo1.84 (Immunochemical Techniques (S
elected Immunoassays (Part D)), ibid. Vol.92 (Immu
nochemical Techniques (Monoclonal Antibodies and G
eneral Immunoassay Methods (PartE)), ibid., Vol. 121
(Immunochemical Techniques (Hybridoma Technology
and Monoclonal Antibodies (Part I) (see Academic Press). As described above, the receptor of the present invention can be quantified with high sensitivity by using the receptor antibody of the present invention.

In the present specification and drawings, when bases, amino acids and the like are indicated by abbreviations, IUPAC-IUB Commisio
Abbreviations based on non-biochemical nomenclature or conventional abbreviations in the art, examples of which are described below. When an amino acid may have an optical isomer, the L-form is indicated 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 triphosphate Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate EIA: Enzyme immunoassay G1y: Glycine A1a: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine G1u: Glutamate Asp: Aspartate Lys: Lysine Arg: Arginine His: Histidine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine G1n: Glue Tamine pGl: Pyroglutamic acid Me: Methyl group Et: Ethyl group Bu: Butyl group Ph: Phenyl group TC: Thiazolidine-4 (R) -carboxamide group

The sequence numbers in the sequence listing in the present specification indicate the following sequences. [SEQ ID NO: 1] This shows the base sequence of a synthetic DNA primer used for amplifying cDNA encoding the receptor protein of the present invention by RT-PCR. [SEQ ID NO: 2] This shows the base sequence of synthetic DNA primer used for amplifying cDNA encoding the receptor protein of the present invention by RT-PCR. [SEQ ID NO: 3] 8 characteristic of the receptor protein of the present invention
Mouse embryonic tumor cells P19 that encode a single amino acid sequence
1 shows the nucleotide sequence of the cDNA derived from it. [SEQ ID NO: 4] This shows the base sequence of cDNA encoding the receptor protein of the present invention. [SEQ ID NO: 5] This shows the amino acid sequence of the receptor protein of the present invention. [SEQ ID NO: 6] 8 characteristic of the receptor protein of the present invention
1 shows the nucleotide sequence of a cDNA derived from a frog embryo, which encodes the amino acid sequences of the present invention. [SEQ ID NO: 7] 8 characteristic of the receptor protein of the present invention
1 shows the amino acid sequence of the amino acid sequence. [SEQ ID NO: 8] Synthetic D of SEQ ID NO: 1 and SEQ ID NO: 2 from mouse embryonic tumor cell line P19 induced by retinoic acid
The base sequence of a DNA fragment amplified by the RT-PCR method using NA is shown. [SEQ ID NO: 9] This shows the base sequence of synthetic DNA primer used for amplifying cDNA encoding the receptor protein of the present invention by RT-PCR. [SEQ ID NO: 10] This shows the base sequence of synthetic DNA primer used for amplifying cDNA encoding the receptor protein of the present invention by RT-PCR.

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the scope of the present invention.

[0054]

【Example】

Example 1 Preparation of po1y (A) ⁺ RNA Fraction from Mouse Embryonic Tumor Cell Line P19 Induced by Retinoic Acid Neurogenic mouse embryonic tumor cell ECP19 cells were cultured at 10 ⁵ cells / ml containing 10% bovine serum α- Seed in a dish for E. coli with minimum essential medium, and retinoic acid (all-trans;
a) (5 × 10 ⁻⁷ M), and then added at 37 ° C., 5% CO ₂ ,
The suspension culture was performed for 72 hours in ir. Thereafter, the cells were washed with the same medium, retinoic acid was removed, the cells were transferred to a tissue culture dish, and the cells were further cultured for 3 days. After culturing, the cells are collected by centrifugation, and guanidine isothiocyanate
After preparing RNA (Kaplan BBet a1., Biochem. J. 183, 1
81-184 (1979)) and an mRNA purification kit (Pharmacia) to prepare a po1y (A) ⁺ RNA fraction.

Example 2 RT-PCR Using Poly (A) ⁺ RNA Derived from Mouse Embryonic Tumor Cells Induced by Retinoic Acid
Amplification of cDNA by Method Using 1 μg of poly (A) ⁺ RNA prepared from a mouse embryonic tumor cell line P19 in which nerve induction was performed with retinoic acid in Example 1, amplification was performed by RT-PCR. The reaction was carried out using synthetic DNA primers (SEQ ID NO: 1 and SEQ ID NO: 2) at 100 pM each and using TaKaRa RNA PCR kit (ver. 2) (manufactured by Takara Shuzo) according to the recipe. Cycle for amplification is thermal cycler (Perkin-Elmer)
A cycle of 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1.5 minutes was repeated 30 times. Confirmation of the amplification product was performed by 2.0% agarose gel electrophoresis and ethidium bromide staining.

Example 3 Subcloning of RT-PCR Product into a Plasmid Vector and Selection of a New Receptor Complementary Clone by Decoding the Base Sequence of the Inserted cDNA Part The reaction product after RT-PCR performed in Example 2 was 2.0%. Separation was performed using a low-melting point agarose gel, and the band was cut out with a razor, followed by heat melting, phenol extraction, and ethanol precipitation to recover DNA. The recovered DNA was subcloned into a plasmid vector according to the prescription of a TA cloning kit (Invitrogen). This was introduced into Escherichia coli JM109 competent cell (manufactured by Takara Shuzo Co., Ltd.) and transformed, and the clone having the cDNA insert was cloned into ampicillin, IPTG (Isopropyl thio-β-D-galactoside) and X-gal (5-Bromo-4). -chloro-3-indolyl-β-D-galactos
ide), and only white-colored clones were separated using a sterilized fingernail to obtain a transformant of Escherichia coli JM109. Each clone was cultured overnight in an LB medium containing ampicillin, and plasmid DNA was prepared using an automatic plasmid extractor (Kurabo). A portion of the prepared DNA was digested with EcoRI to confirm the size of the inserted cDNA fragment. A part of the remaining DNA was further treated with RNase, extracted with phenol / chloroform, concentrated by ethanol precipitation, and used for determination of the following nucleotide sequence. The reaction for base sequence determination is Dye Deoxy Terminator Cyc
This was performed using a le Sequencing Kit (manufactured by ABI), and decoding was performed using a fluorescent automatic sequencer. Obtained nucleotide sequence information is DNASIS (Hitachi System Engineering Co., Ltd.)
This was performed using The determined nucleotide sequence is shown in SEQ ID NO: 8. A homology search was performed based on the determined base sequence. As a result, Escherichia coli
C inserted into the plasmid carried by the transformant of JM109
The DNA fragment was found to encode a novel receptor protein in which the 24-base pair nucleotide sequence shown in SEQ ID NO: 3 was inserted into a known mouse activin receptor.

Example 4 Cloning of cDNA Containing the Entire Coding Region of Receptor Protein from cDNA Library Derived from Mouse Embryonic Tumor Cell Line P19 Induced by Retinoic Acid Example 1 Mice Induced by Retinoic Acid Using 10 μg of poly (A) + RNA prepared from embryonic tumor cells P19, cDN
Follow the recipe of A Synthesis System P1us (manufactured by Amasyamu) with first-strand random 9mer primer.
cDNA was synthesized. Subsequently, a second-strand cDNA was synthesized to obtain 269 ng of a double-strand cDNA. These are cDN
A rapid adaptor ligation module (Amasiyam)
And an adapter was ligated to both ends of the double-strand cDNA previously synthesized according to the manual. In addition, these were added to λZAP II / Eco RI / CIAP Treated Vecto
Using r Kit (manufactured by Toyobo), a vector arm of λZAP II was ligated to 75 ng of double-stranded cDNA according to the prescription. A cDNA library of 2.1 × 10 ⁶ pfu (plaque forming unit) was mixed with Escherichia coli XL1-BlueMRF ′ treated with magnesium sulfate.
After incubating at 7 ° C. for 15 minutes, 0.5% agarose LB was added, and the resultant was plated on an 1.5% agar (Wako Pure Chemical Industries) LB plate. A nitrocellulose filter was placed on the plate with the plaque, and the plaque was transferred onto the filter. After denaturing this filter by alkali treatment,
The DNA was fixed by heating at 80 ° C for 3 hours. Filter this filter with 50% formamide, 5 × SSPE (SSPE; 150 mM N
_{aC1,10mM NaH 2 P0 4 · H 2} 0,1.25mM EDTA pH7.4), 5 × Den
hardt's solution, 0.1% SDS (Sodium dodecyl sulfate), 1
Hybridization was carried out by incubating overnight at 42 ° C. with a probe described below in a buffer containing 00 μg / ml salmon sperm DNA at 42 ° C. As a probe, the DNA fragment inserted into the plasmid obtained in Example 3 was cleaved with EcoRI, recovered, and then used with a random primed DNA labeling kit (manufactured by Amersham) using [ ³² P] dCTP (manufactured by DuPont). ) Was used by labeling. Washing, 2 × SSC (SSC; 150mM NaC1,10mM NaH 2 P0 4 · H 2 0),
The reaction was performed with 0.1% SDS at room temperature for 1 hour and 15 minutes, then at 60 ° C. for 1 hour and 15 minutes, and then autoradiography was performed at −80 ° C. to detect hybridizing plaques. As a result of the screening, hybridization signals were observed in 30 independent plaques. Poke these and open them, 5 ml each of SM (50 mM Tris-HC1, pH 7.5, 0.1 MN
aCl, 7 mM MgSO ₄ 0.01% gelatin), stirred well, and partially treated with magnesium sulfate.
Mix with BlueMRF ', incubate at 37 ° C for 15 minutes, add 0.5% agarose (Pharmacia) LB,
The seeds were seeded on 1.5% agar (Wako Pure Chemical Industries) LB plate. Nylon filters were placed on these plates and plaques were transferred onto the filters. This filter is denatured with alkali in the same manner as described above, and then heated at 80 ° C for 3 hours.
Fixation of NA was performed. These filters were hybridized using the same probe in the same manner as described above, and washed with the same washing method to detect hybridizing plaques. This screen revealed that 28 clones were positive. Therefore, in order to determine whether these clones encode a known activin receptor protein or a novel receptor protein according to the present invention, individual plaques were suspended in distilled water, and this was suspended in a template. DNA, and PCR was performed using the synthetic DNAs shown in SEQ ID NO: 1 and SEQ ID NO: 2 as primers,
The length of the DNA fragment to be amplified was confirmed. This screening revealed that three clones encoded the novel receptor protein. Therefore, the insert of this clone was cut out with Eco RI, and the fragment was pUC118
After subcloning into the Eco RI site of E. coli, Escherichia coli JM109 was transformed with this plasmid to obtain a transformant.
Next, the nucleotide sequence of the cDNA fragment inserted into the plasmid was determined. Specifically, an unnecessary portion was removed or a necessary fragment was subcloned using a restriction enzyme site present in the EcoRI fragment to prepare a template plasmid for nucleotide sequence analysis. The reaction for sequencing is
Using the Dye Deoxy Terminator Cycle Sequencing Kit (ABI), a fluorescent DNA sequencer (ABI)
And DNASIS (Hitachi Software Engineering) was used for data analysis. The determined base sequence is shown in FIGS. This nucleotide sequence is
vol.65, pp.973-982, 1991. It completely matches the mouse activin receptor type IIA except for the nucleotide sequence shown in SEQ ID NO: 3, and is a new subtype of the mouse activin receptor type IIA. It was shown that.

Example 5 Poly (A) ⁺ R derived from various organs of mouse
Detection of expression by RT-PCR using NA Ovarian, testis, whole embryo, lung, heart, liver, skeletal muscle,
Amplification by RT-PCR was performed using 1 μg of poly (A) ⁺ RNA prepared from the brain as a template. The reaction was performed using synthetic DNA primers (SEQ ID NO: 1 and SEQ ID NO: 2) at 100 pM each.
Using TaKaRa RNA PCR kit (ver.2) (Takara Shuzo) according to the recipe. Cycle for amplification is thermal cycler (Perkin-Elmer)
A cycle of 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1.5 minutes was repeated 30 times. Confirmation of the amplification product was performed by 2.0% agarose gel electrophoresis and ethidium bromide staining. The result is shown in FIG. The expression of the receptor of the present invention was specifically confirmed in the brain, similarly to mouse embryonic tumor cells P19 induced by retinoic acid. From this, it was confirmed that the receptor of the present invention was specifically expressed in the nervous system.

Example 6 RT-PC using human neuroblastoma-derived cell line or frog embryo-derived poly (A) ⁺ RNA
Amplification of cDNA by R method Using commercially available human neuroblastoma-derived poly (A) ⁺ RNA (manufactured by Toyobo Co., Ltd.) or frog embryo-derived poly (A) ⁺ RNA 1 μg as template, amplification was performed by RT-PCR. . The reaction was performed using TaKaRa RNA PCR kit (ver. 2) using synthetic DNA primers (human: SEQ ID NO: 1 and SEQ ID NO: 2, frog: SEQ ID NO: 9 and SEQ ID NO: 10) at 100 pM each.
(Manufactured by Takara Shuzo) according to the prescription. The cycle for amplification was 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1.
The 5 minute cycle was repeated 30 times. Confirmation of amplification product is 2.0
% Agarose gel electrophoresis and ethidium bromide staining.

Example 7 Subcloning of RT-PCR Product into a Plasmid Vector and Selection of a Novel Receptor Complementary Clone by Decoding the Base Sequence of the Inserted cDNA Part The reaction product after RT-PCR performed in Example 6 was 1.0%. Separation was performed using a low-melting point agarose gel, and the band was cut out with a razor, followed by heat melting, phenol extraction, and ethanol precipitation to recover DNA. The recovered DNA was subcloned into a plasmid vector according to the prescription of a TA cloning kit (Invitrogen). This was introduced into Escherichia coli JM109 competent cell (manufactured by Takara Shuzo Co., Ltd.) and transformed, and the clone having the cDNA insert was cloned into ampicillin, IPTG (Isopropyl thio-β-D-galactoside) and X-gal (5-Bromo-4). -chloro-3-indolyl-β-D-galact
oside), and only clones exhibiting white color were separated using sterilized nail picking to obtain a transformant, Escherichia coli. Individual clones were cultured overnight in LB medium containing ampicillin,
Plasmid DNA was prepared using an automatic plasmid extraction device (manufactured by Kurabo Industries, Ltd.) and subjected to the following nucleotide sequence determination. The reaction for base sequence determination is Dye Deoxy Terminator Cyc
This was performed using a le Sequencing Kit (manufactured by ABI), and decoding was performed using a fluorescent automatic sequencer. Obtained nucleotide sequence information is DNASIS (Hitachi System Engineering Co., Ltd.)
This was performed using As a result of a homology search based on the determined base sequence, the cDNA fragment derived from the human neuroblastoma-derived cell line was identical to SEQ ID NO: 3. The amino acid sequence deduced from this nucleotide sequence is SEQ ID NO: 7
Indicated by The nucleotide sequence of the frog embryo-derived cDNA fragment was represented by SEQ ID NO: 6, and the amino acid sequence deduced therefrom was identical to SEQ ID NO: 7.

[0061]

EFFECTS OF THE INVENTION The receptor protein of the present invention and the DNA encoding the protein can be used to determine ligands, obtain antibodies and serum, construct a recombinant receptor protein expression system, and develop a receptor binding assay system using the expression system. Screening of drug candidate compounds, implementation of drug design based on comparison with structurally similar ligands / receptors, probes for gene diagnosis,
It can be used as a reagent in the preparation of PCR primers and the like, and can also be used as a drug for gene therapy and the like. In particular, elucidation of the structure and properties of activin receptors will lead to the development of unique drugs acting on these systems.

[0062]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: TACCCTCCTGTACTTGTTCCTACTCAA SEQ ID NO: 2 Sequence length: 27 Sequence Type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: TAGCCACAGGTCCACATCCACACTGGT SEQ ID NO: 3 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Double strand Topology: linear Sequence type: cDNA sequence: CACGCCTTTCATATAATGATAGAG

SEQ ID NO: 4 Sequence length: 2122 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence: TCGGGAAA 8 ATG GGA GCT GCT GCA AAG TTG GCG TTC GCC GTC TTT CTT ATC TCT TGC 56 Met Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys 1 5 10 15 TCT TCA GGT GCT ATA CTT GGC AGA TCA GAA ACT CAG GAG TGT CTT TTC 104 Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe 20 25 30 TTT AAT GCT AAT TGG GAA AGA GAC AGA ACC AAC CAG ACT GGT GTT GAA 152 Phe Asn Ala Asn Trp Glu Arg Asp Arg Thr Asn Gln Thr Gly Val Glu 35 40 45 CCT TGC TAT GGT GAT AAA GAT AAA CGG CGA CAT TGT TTT GCT ACC TGG 200 Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp 50 55 60 AAG AAT ATT TCT GGT TCC ATT GAA ATA GTG AAG CAA GGT TGT TGG CTG 248 Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu 65 70 75 80 GAT GAT ATC AAC TGC TAT GAC AGG ACT GAT TGT ATA GAA AAA AAA GAC 296 Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Ile Gl u Lys Lys Asp 85 90 95 AGC CCT GAA GTG TAC TTT TGT TGC TGT GAG GGC AAT ATG TGT AAT GAA 344 Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu 100 105 110 AAG TTC TCT TAT TTT CCG GAG ATG GAA GTC ACA CAG CCC ACT TCA AAT 392 Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn 115 120 125 CCT GTT ACA CCG AAG CCA CCC TAT TAC AAC ATT CTG CTG TAT TCC TTG 440 Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu 130 135 140 GTA CCA CTA ATG TTA ATT GCA GGA ATT GTC ATT TGT GCA TTT TGG GTG 488 Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile Cys Ala Phe Trp Val 145 150 155 160 TAC AGA CAT CAC AAG ATG GCC TAC CCT CCT GTA CTT GTT CCT ACT CAA 536 Tyr Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gln 165 170 175 CAC GCC TTT CAT ATA ATG ATA GAG GAC CCA GGA CCA CCC CCA CCT TCC 584 His Ala Phe His Ile Met Ile Glu Asp Pro Gly Pro Pro Pro Pro Ser 180 185 190 CCA TTA CTA GGG TTG AAG CCA TTG CAG CTG TTA GAA GTG AAA GCA AGG 632 Pro Leu Leu Gly Leu Lys Pro Leu Gln Leu L eu Glu Val Lys Ala Arg 195 200 205 GGA AGA TTT GGT TGT GTC TGG AAA GCC CAG TTG CTC AAT GAA TAT GTG 680 Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Leu Asn Glu Tyr Val 210 215 220 GCT GTC AAA ATA TTT CCA ATA CAG GAC AAA CAG TCC TGG CAG AAT GAA 728 Ala Val Lys Ile Phe Pro Ile Gln Asp Lys Gln Ser Trp Gln Asn Glu 225 230 235 240 TAT GAA GTC TAT AGT CTA CCT GGA ATG AAG CAT GAG AAC ATA CTA CAG 776 Tyr Glu Val Tyr Ser Leu Pro Gly Met Lys His Glu Asn Ile Leu Gln 245 250 255 TTC ATT GGT GCA GAG AAA AGA GGC ACC AGT GTG GAT GTG GAC CTG TGG 824 Phe Ile Gly Ala Glu Lys Arg Gly Thr Ser Val Asp Val Asp Leu Trp 260 265 270 CTA ATC ACA GCA TTT CAT GAA AAG GGC TCA CTG TCA GAC TTT CTT AAG 872 Leu Ile Thr Ala Phe His Glu Lys Gly Ser Leu Ser Asp Phe Leu Lys 275 280 285 285 GCT AAT GTG GTC TCT TGG AAT GAA CTT TGT CAT ATT GCA GAA ACC ATG 920 Ala Asn Val Val Ser Trp Asn Glu Leu Cys His Ile Ala Glu Thr Met 290 295 300 GCT AGA GGA TTG GCA TAT TTA CAT GAG GAT ATA CCT GGC TTA AAA GAT 968 Ala Arg Gly Leu Ala Tyr Leu H is Glu Asp Ile Pro Gly Leu Lys Asp 305 310 315 320 GGC CAC AAG CCT GCA ATC TCT CAC AGG GAC ATC AAA AGT AAA AAT GTG 1016 Gly His Lys Pro Ala Ile Ser His Arg Asp Ile Lys Ser Lys Asn Val 325 330 335 CTG TTG AAA AAC AAT CTG ACA GCT TGC ATT GCT GAC TTT GGG TTG GCC 1064 Leu Leu Lys Asn Asn Leu Thr Ala Cys Ile Ala Asp Phe Gly Leu Ala 340 345 350 TTA AAG TTC GAG GCT GGC AAG TCT GCA GGT GAC ACC CAT GGG CAG GTT 1112 Leu Lys Phe Glu Ala Gly Lys Ser Ala Gly Asp Thr His Gly Gln Val 355 360 365 GGT ACC CGG AGG TAT ATG GCT CCA GAG GTG TTG GAG GGT GCT ATA AAC 1160 Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn 370 375 380 TTC CAA AGG GAC GCA TTT CTG AGG ATA GAT ATG TAC GCC ATG GGA TTA 1208 Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu 385 390 395 395 400 GTC CTA TGG GATGTG TCT CGT TGC ACT GCT GCA GAT GGA CCC GTA 1256 Val Leu Trp Glu Leu Ala Ser Arg Cys Thr Ala Ala Asp Gly Pro Val 405 410 415 GAT GAG TAC ATG TTA CCA TTT GAG GAA GAA ATT GGC CAG CAT CCA TCT 1304 Asp Gl u Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser 420 425 430 CTT GAA GAT ATG CAG GAA GTT GTT GTG CAT AAA AAA AAG AGG CCT GTT 1352 Leu Glu Asp Met Gln Glu Val Val Val His Lys Lys Lys Arg Pro Val 435 440 445 TTA AGA GAT TAT TGG CAG AAA CAT GCA GGA ATG GCA ATG CTC TGT GAA 1400 Leu Arg Asp Tyr Trp Gln Lys His Ala Gly Met Ala Met Leu Cys Glu 450 455 460 ACG ATA GAA GAA TGT TGG GAT CAT GAT GCA GAA GCC AGG TTA TCA GCT 1448 Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala 465 470 475 480 GGA TGT GTA GGT GAA AGA ATT ACT CAG ATG CAA AGA CTA ACA AAT ATC 1496 Gly Cys Val Gly Glu Arg Ile Thr Gln Met Gln Arg Leu Thr Asn Ile 485 490 495 ATT ACT ACA GAG GAC ATT GTA ACA GTG GTC ACA ATG GTG ACA AAT GTT 1544 Ile Thr Thr Glu Asp Ile Val Thr Val Val Thr Met Val Thr Asn Val 500 505 510 GAC TTT CCT CCC AAA GAA TCT AGT CTA TGATGGTGGC ACCGTCTGTA 1591 Asp Phe Pro Pro Lys Glu Ser Ser Leu 515 520 521 CACACTGAGG ACTGGGACTC TGAACTGGAG CTGCTAAGCT AAGGAAAGTG CTTAGTTGAT 1651 TTTCTGTGTG AAATGAGT AG GATGCCTCCA GGACATGTAC GCAAGCAGCC CCTTGTGGAA 1711 AGCATGGATC TGGGAGATGG ATCTGGGAAA CTTACTGCAT CGTCTGCAGC ACAGATATGA 1771 AGAGGAGTCT AAGGGAAAAG CTGCAAACTG TAAAGAACTT CTGAAAATGT ACTCGAAGAA 1831 TGTGGCCCTC TCCAAATCAA GGATCTTTTG GACCTGGCTA ATCAAGTATT TGCAAAACTG 1891 ACATCAGATT TCTTAATGTC TGTCAGAAGA CACTAATTCC TTAAATGAAC TACTGCTATT 1951 TTTTTTAAAT GAAAAACTTT TCATTTCAGA TTTTAAAAAG GGTAACTTTT TATTGCATTT 2011 GCTGTTGTTT CTATAAATGA CTATTGTAAT GCCAACATGA CACAGCTTGT GAATGTGTAG 2071 TGTGCTGCTG TTCTGTGTAC ATAGTCATCA AAGTGGGGTA CAGTAAAGAG G 2122

SEQ ID NO: 5 Sequence length: 521 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence: Met Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys 1 5 10 15 Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe 20 25 30 Phe Asn Ala Asn Trp Glu Arg Asp Arg Thr Asn Gln Thr Gly Val Glu 35 40 45 Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp 50 55 60 Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu 65 70 75 80 Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Ile Glu Lys Lys Asp 85 90 95 Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu 100 105 110 Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn 115 120 125 Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu 130 135 140 Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile Cys Ala Phe Trp Val 145 150 155 160 Tyr Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gln 165 170 175 His Ala Phe His Ile M et Ile Glu Asp Pro Gly Pro Pro Pro Pro Ser 180 185 190 Pro Leu Leu Gly Leu Lys Pro Leu Gln Leu Leu Glu Val Lys Ala Arg 195 200 205 Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Leu Asn Glu Tyr Val 210 215 220 Ala Val Lys Ile Phe Pro Ile Gln Asp Lys Gln Ser Trp Gln Asn Glu 225 230 235 240 Tyr Glu Val Tyr Ser Leu Pro Gly Met Lys His Glu Asn Ile Leu Gln 245 250 255 Phe Ile Gly Ala Glu Lys Arg Gly Thr Ser Val Asp Val Asp Leu Trp 260 265 270 Leu Ile Thr Ala Phe His Glu Lys Gly Ser Leu Ser Asp Phe Leu Lys 275 280 285 Ala Asn Val Val Ser Trp Asn Glu Leu Cys His Ile Ala Glu Thr Met 290 295 300 Ala Arg Gly Leu Ala Tyr Leu His Glu Asp Ile Pro Gly Leu Lys Asp 305 310 315 320 Gly His Lys Pro Ala Ile Ser His Arg Asp Ile Lys Ser Lys Asn Val 325 330 335 Leu Leu Lys Asn Asn Leu Thr Ala Cys Ile Ala Asp Phe Gly Leu Ala 340 345 350 Leu Lys Phe Glu Ala Gly Lys Ser Ala Gly Asp Thr His Gly Gln Val 355 360 365 Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn 370 375 380 380 Phe Gln Arg Asp Ala Phe L eu Arg Ile Asp Met Tyr Ala Met Gly Leu 385 390 395 400 400 Val Leu Trp Glu Leu Ala Ser Arg Cys Thr Ala Ala Asp Gly Pro Val 405 410 415 Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser 420 425 430 Leu Glu Asp Met Gln Glu Val Val Val His Lys Lys Lys Arg Pro Val 435 440 445 Leu Arg Asp Tyr Trp Gln Lys His Ala Gly Met Ala Met Leu Cys Glu 450 455 460 Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala 465 470 475 480 480 Gly Cys Val Gly Glu Arg Ile Thr Gln Met Gln Arg Leu Thr Asn Ile 485 490 495 Ile Thr Thr Glu Asp Ile Val Thr Val Val Thr Thr Met Val Thr Asn Val 500 505 510 Asp Phe Pro Pro Lys Glu Ser Ser Leu 515 520 521

SEQ ID NO: 6 Sequence length: 24 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA sequence: CACGCCTTTCACATTATGATAGAG SEQ ID NO: 7 Sequence length: 8 Type: Amino acid Topology: Linear Sequence type: Peptide Sequence: His Ala Phe His Ile Met Ile Glu SEQ ID NO: 8 Sequence length: 294 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear sequence type: cDNA sequence: TACCCTCCTG TACTTGTTCC TACTCAAGAC CCAGGACCAC CCCCACCTTC CCCATTACTA 60 GGGTTGAAGC CATTGCAGCT GTTAGAAGTG AAAGCAAGGG GAAGATTTGG TTGTGTCTGG 120 AAAGCCCAGT TGCTCAATGA ATATGTGGCT GTCAAAATAT TTCCAATACA GGACAAACAG 180 TCCTGGCAGA ATGAATATGA AGTCTATAGT CTACCTGGAA TGAAGCATGA GAACATACTA 240 CAGTTCATTG GTGCAGAGAA AAGAGGCACC AGTGTGGATG TGGACCTGTG GCTA 294

SEQ ID NO: 9 Sequence length: 19 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: TCGCCTACCCCCCAGTGCT SEQ ID NO: 10 Sequence length : 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: TCTGTGTCAAGGTTTGT

[0067]

[Brief description of the drawings]

FIG. 1. Mouse activin receptor type IIA (A
TR) The entire base sequence of the cDNA encoding the protein and the first half of the deduced amino acid sequence. The underlined part (single line)
The position of the synthetic primer used in RT-PCR is shown. (Continued from Fig. 2)

[Fig. 2] Mouse activin receptor type IIA (A
TR) The entire nucleotide sequence of the cDNA encoding the protein and the latter half of the deduced amino acid sequence. (Continuation of Fig. 1)

[Fig. 3] A novel receptor c obtained by RT-PCR from mouse embryonic tumor cell line P19 induced by retinoic acid.
The first half of the entire nucleotide sequence of DNA. (Continued from FIG. 4)

FIG. 4 shows a novel receptor c obtained by RT-PCR from a mouse embryonic tumor cell line P19 induced by retinoic acid.
The latter half of the entire DNA sequence. (Continuation of FIG. 3)

FIG. 5 is the first half of the amino acid sequence deduced from the nucleotide sequences shown in FIGS. 3 and 4; (Continued from FIG. 6)

FIG. 6 is the first half of the amino acid sequence deduced from the nucleotide sequences shown in FIGS. 3 and 4; (Continuation of FIG. 5)

[FIG. 7] Analysis of expression by RT-PCR using poly (A) ⁺ RNA derived from various mouse organs or mouse embryonic tumor cell line P19 induced by retinoic acid. (See Example 5) ATR in the figure indicates an RT-PCR product corresponding to the known mouse activin receptor type IIA, and ATR IIA-N indicates an RT-PCR product corresponding to the novel receptor of the present invention.

FIG. 8 shows the first half of the entire nucleotide sequence of the cDNA encoding the frog activin receptor type II protein. (Continued from FIG. 9)

FIG. 9 is the latter half of the entire nucleotide sequence of the cDNA encoding the frog activin receptor type II protein. (Continuation of FIG. 8)

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication location G01N 33/53 G01N 33/566 33/566 9282-4B C12N 15/00 ZNAA // A61K 38/00 A61K 37/02

Claims (22)

[Claims] 1. A receptor protein having an amino acid sequence substantially identical to the amino acid sequence represented by the formula His Ala Phe His Ile Met Ile Glu, or a salt thereof. 2. The receptor protein according to claim 1, wherein the receptor protein is a neural cell-specific receptor protein. 3. The receptor protein according to claim 2, wherein the receptor protein is an activin receptor protein. 4. An amino acid sequence substantially identical to the amino acid sequence represented by the formula His Ala Phe His Ile Met Ile Glu, wherein the formula X-Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn Trp Glu Arg Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Ile Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile Cys Ala Phe Trp Val Tyr Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gln His Ala Phe His Ile Met Ile Glu Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Leu Gly Leu Lys Pro Leu Gln Leu Leu Glu Val Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Leu Asn Glu Tyr Val Ala Val Lys Ile Phe Pro Ile Gln Asp Lys Gln Ser Trp Gln Asn Glu Tyr Glu Val Tyr Ser Leu Pro Gly Met Lys His Glu Asn Ile Leu Gln Phe Ile Gly Ala Glu Lys Arg Gly Thr Ser Val Asp Val Asp Leu Trp Leu Ile Thr Ala Phe His Glu Lys Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val Ser Trp Asn Glu Leu Cys His Ile Ala Glu Thr Met Ala Arg Gly Leu Ala Tyr Leu His Glu Asp Ile Pro Gly Leu Lys Asp Gly His Lys Pro Ala Ile Ser His Arg Asp Ile Lys Ser Lys Asn Val Leu Leu Lys Asn Asn Leu Thr Ala Cys Ile Ala Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala Gly Lys Ser Ala Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Ala Ser Arg Cys Thr Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Asp Met Gln Glu Val Val Val His Lys Lys Lys Arg Pro Val Leu Arg Asp Tyr Trp Gln Lys His Ala Gly Met Ala Met Leu Cys Glu Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Gly Glu Arg Ile Thr Gln Met Gln Arg Leu Thr Asn Ile Ile Thr Thr Glu Asp Ile Val Thr Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys Glu Ser Ser Leu wherein X represents optionally protected Met or hydrogen. Or a salt thereof, comprising an amino acid sequence represented by the formula: The formula X-Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn Trp Glu Arg Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Ile Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile Cys Ala Phe Trp Val Tyr Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gln His Ala Phe His Ile Met Ile Glu Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Leu Gly Leu Lys Pro Leu Gln Leu Leu Glu Val Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Leu Asn Glu Tyr Val Ala Val Lys Ile Phe Pro Ile Gln Asp Lys Gln Ser Trp Gln Asn Glu Tyr Glu Val Tyr S er Leu Pro Gly Met Lys His Glu Asn Ile Leu Gln Phe Ile Gly Ala Glu Lys Arg Gly Thr Ser Val Asp Val Asp Leu Trp Leu Ile Thr Ala Phe His Glu Lys Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val Ser Trp Asn Glu Leu Cys His Ile Ala Glu Thr Met Ala Arg Gly Leu Ala Tyr Leu His Glu Asp Ile Pro Gly Leu Lys Asp Gly His Lys Pro Ala Ile Ser His Arg Asp Ile Lys Ser Lys Asn Val Leu Leu Lys Asn Asn Leu Thr Ala Cys Ile Ala Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala Gly Lys Ser Ala Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Ala Ser Arg Cys Thr Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Asp Met Gln Glu Val Val Val His Lys Lys Lys Arg Pro Val Leu Arg Asp Tyr Trp Gln Lys His Ala Gly Met Ala Met Leu Cys Glu Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Gly Glu Arg Ile Thr Gln Met Gln Arg Leu Thr A sn Ile Ile Thr Thr Glu Asp Ile Val Thr Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys Glu Ser Ser Leu [where X represents Met or hydrogen which may be protected. In the amino acid sequence represented by the formula, one or more amino acids other than the amino acid sequence represented by the formula His Ala Phe His Ile Met Ile Glu may be deleted, added, inserted or substituted. Or a salt thereof. The formula X-Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu Ile Ser Cys Ser Ser Gly Ala Ile Leu Gly Arg Ser Glu Thr Gln Glu Cys Leu Phe Phe Asn Ala Asn Trp Glu Arg Asp Arg Thr Asn Gln Thr Gly Val Glu Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile Val Lys Gln Gly Cys Trp Leu Asp Asp Ile Asn Cys Tyr Asp Arg Thr Asp Cys Ile Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gln Pro Thr Ser Asn Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn Ile Leu Leu Tyr Ser Leu Val Pro Leu Met Leu Ile Ala Gly Ile Val Ile Cys Ala Phe Trp Val Tyr Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gln His Ala Phe His Ile Met Ile Glu Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Leu Gly Leu Lys Pro Leu Gln Leu Leu Glu Val Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln Leu Leu Asn Glu Tyr Val Ala Val Lys Ile Phe Pro Ile Gln Asp Lys Gln Ser Trp Gln Asn Glu Tyr Glu Val Tyr S er Leu Pro Gly Met Lys His Glu Asn Ile Leu Gln Phe Ile Gly Ala Glu Lys Arg Gly Thr Ser Val Asp Val Asp Leu Trp Leu Ile Thr Ala Phe His Glu Lys Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val Ser Trp Asn Glu Leu Cys His Ile Ala Glu Thr Met Ala Arg Gly Leu Ala Tyr Leu His Glu Asp Ile Pro Gly Leu Lys Asp Gly His Lys Pro Ala Ile Ser His Arg Asp Ile Lys Ser Lys Asn Val Leu Leu Lys Asn Asn Leu Thr Ala Cys Ile Ala Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala Gly Lys Ser Ala Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp Ala Phe Leu Arg Ile Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Ala Ser Arg Cys Thr Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu Glu Glu Ile Gly Gln His Pro Ser Leu Glu Asp Met Gln Glu Val Val Val His Lys Lys Lys Arg Pro Val Leu Arg Asp Tyr Trp Gln Lys His Ala Gly Met Ala Met Leu Cys Glu Thr Ile Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Gly Glu Arg Ile Thr Gln Met Gln Arg Leu Thr A sn Ile Ile Thr Thr Glu Asp Ile Val Thr Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys Glu Ser Ser Leu [where X represents Met or hydrogen which may be protected. The protein or a salt thereof according to claim 1, which comprises an amino acid sequence represented by the formula: 7. A protein comprising an amino acid sequence substantially identical to the amino acid sequence represented by the formula His Ala Phe His Ile Met Ile Glu, and having the ability to bind to activin or a protein substantially identical to activin, or Its salt. 8. A DNA comprising a nucleotide sequence encoding the protein according to claim 1. 9. A DNA comprising a nucleotide sequence encoding the protein according to claim 7. Claim 10: Formula X'GGA GCT GCT GCA AAG TTG GCG TTC GCC GTC TTT CTT ATC TCT TGC TCT TCA GGT GCT ATA CTT GGC AGA TCA GAA ACT CAG GAG TGT CTT TTC TTT AAT GCT AAT TGG GAA AGA GAC AGA ACC AAC CAG ACT GGT GTT GAA CCT TGC TAT GGT GAT AAA GAT AAA CGG CGA CAT TGT TTT GCT ACC TGG AAG AAT ATT TCT GGT TCC ATT GAA ATA GTG AAG CAA GGT TGT TGG CTG GAT GAT ATC AAC TGC TAT GAC AGG ACT GAT TGT ATA GA AAA AAA GAC AGC CCT GAA GTG TAC TTT TGT TGC TGT GAG GGC AAT ATG TGT AAT GAA AAG TTC TCT TAT TTT CCG GAG ATG GAA GTC ACA CAG CCC ACT TCA AAT CCT GTT ACA CCG AAG CCA CCC TAT TAC AAC ATT CTG CTG TCC TTG GTA CCA CTA ATG TTA ATT GCA GGA ATT GTC ATT TGT GCA TTT TGG GTG TAC AGA CAT CAC AAG ATG GCC TAC CCT CCT GTA CTT GTT CCT ACT CAA CAC GCC TTT CAT ATA ATG ATA GAG GAC CCA GGA CCA CCC CCA CCC TCC TTA CTA GGG TTG AAG CCA TTG CAG CTG TTA GAA GTG AAA GCA AGG GGA AGA TTT GGT TGT GTC TGG AAA GCC CAG TTG CTC AAT GAA TAT GTG GCT GTC AAA ATA TTT CCA ATA CAG GAC AAA CAG TCC TGG CAG AAT GAA TAT GAA GTC TA T AGT CTA CCT GGA ATG AAG CAT GAG AAC ATA CTA CAG TTC ATT GGT GCA GAG AAA AGA GGC ACC AGT GTG GAT GTG GAC CTG TGG CTA ATC ACA GCA TTT CAT GAA AAG GGC TCA CTG TCA GAC TTT CTT AAG GCT TAT GTG GTC TGG AAT GAA CTT TGT CAT ATT GCA GAA ACC ATG GCT AGA GGA TTG GCA TAT TTA CAT GAG GAT ATA CCT GGC TTA AAA GAT GGC CAC AAG CCT GCA ATC TCT CAC AGG GAC ATC AAA AGT AAA AAT GTG CTG TTG AAA AAC AAT CTG ACA GCT TGC ATT GCT GAC TTT GGG TTG GCC TTA AAG TTC GAG GCT GGC AAG TCT GCA GGT GAC ACC CAT GGG CAG GTT GGT ACC CGG AGG TAT ATG GCT CCA GAG GTG TTG GAG GGT GCT ATA AAC TTC CAA AGG GAC GCA TTT CGG AGG GAT ATG TAC GCC ATG GGA TTA GTC CTA TGG GAA TTG GCT TCT CGT TGC ACT GCT GCA GAT GGA CCC GTA GAT GAG TAC ATG TTA CCA TTT GAG GAA GAA ATT GGC CAG CAT CCA TCT CTT GAA GAT ATG CAG GAA GTT GTT GTG CAT AAA AAG AGG CCT GTT TTA AGA GAT TAT TGG CAG AAA CAT GCA GGA ATG GCA ATG CTC TGT GAA ACG ATA GAA GAA TGT TGG GAT CAT GAT GCA GAA GCC AGG TTA TCA GCT GGA TGT GTA GGT GAA AGA ATT ACT CAG ATG CATAGA AC A AAT ATC ATT ACT ACA GAG GAC ATT GTA ACA GTG GTC ACA ATG GTG ACA AAT GTT GAC TTT CCT CCC AAA GAA TCT AGT CTA [wherein, X ′ represents a base sequence represented by ATG or hydrogen. 9. The DN according to claim 8, which comprises a base sequence represented by the formula:
A. 11. A vector containing the DNA according to claim 8. A transformant carrying the vector according to claim 11. (13) culturing the transformant according to (12),
A method for producing a receptor protein or a salt thereof, comprising producing and accumulating a receptor protein or a salt thereof in a culture and collecting the receptor protein or a salt thereof. (14) culturing the transformant according to (12),
A method for producing a receptor protein or a salt thereof, comprising producing and accumulating a receptor protein or a salt thereof in a culture supernatant or on a cell membrane of a transformant and collecting the receptor protein or a salt thereof. 15. The DNA detection method according to claim 8, wherein the polymerase chain reaction method is carried out using a synthetic DNA containing the base sequence of the formula TACCCTCCTGTACTTGTTCCTACTCAA or the formula TAGCCACAGGTCCACATCCACACTGGT. 16. An antibody against the receptor protein of claim 1 or a salt thereof. 17. A test solution containing the protein of claim 1 or a salt thereof and a labeled protein or a salt of the protein of claim 1 competitively reacted with the antibody of claim 16. The method for quantifying a protein or a salt thereof according to claim 1, characterized in that: 18. A method for determining a ligand for a protein or a salt thereof according to claim 1, wherein the test compound is brought into contact with the protein or a salt thereof according to claim 1. (19) A case where a ligand or a test compound is brought into contact with the protein or a salt thereof according to the above (1). 2. The method for screening for a compound or a salt thereof that inhibits or promotes the binding of a protein or a salt thereof to a ligand according to claim 1, wherein the comparison is performed. 20. A protein comprising the protein according to claim 1 or a salt thereof and a ligand thereto, wherein the ligand comprises the protein according to claim 1 or a salt thereof, or a known activin receptor protein or a salt thereof. A kit for screening a compound or a salt thereof that inhibits or promotes the binding of a compound. 21. An activin receptor agonist or an activin receptor antagonist obtained by using the screening method according to claim 19 or the screening kit according to claim 20. 22. A method for diagnosing a neurodegenerative disease, comprising using the DNA detection method according to claim 15.