JPH10175998A - Protein related to communication of information - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein having a cysteine-rich domain and an SH3 domain and related to the communication of information in nerve cells. SOLUTION: This protein has a cysteine-rich domain and an SH3 domain. The cysteine-rich domain has an amino acid sequence comprising the amino acid residues 108-159 of the formula or a changed sequence obtained by subjecting one or several amino acid sequences of this amino acid sequence to one or more of substitution, deletion, insertion and addition treatments. The SH3 domain has an amino acid sequence comprising the amino acid residues 292-339 of the formula or a changed sequence obtained by subjecting one or several amino acid sequences of the amino acid sequence to one or more of substitution, deletion, insertion and addition treatments. The protein is obtained by culturing a transformant containing a recombined vector containing a polypeptide molecular coding the protein and subsequently recovering the product from the culture product or the cultured cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel protein, a DNA encoding the same, and a method for producing the same. More specifically, proteins specifically expressed in the brain and involved in brain-specific signal transduction and DNs encoding the same
About A.

[0002]

2. Description of the Related Art Conventionally, a so-called "functional cloning method" has been generally used for obtaining a novel gene. It obtains information on the amino acid sequence of the target protein or information on antibodies or substances that specifically interact with the protein (eg, ligands and receptors), and encodes unknown proteins based on the characteristics of their functions. This is a method of obtaining a gene to be expressed. For example, a gene encoding one of the signaling substances, Grb2 (also referred to as ASH), binds to the substance when 1) the C-terminal side of the EGF receptor present in cells is phosphorylated; and 2) Cloned independently by separate researchers, taking advantage of the two features of having an SH2 domain. That is, based on the property described in 1), a recombinant protein on the C-terminal side of the EGF receptor was prepared, phosphorylated with [γ- ³² P] ATP, and the expression of the λgt11 human brain-derived expression was performed using this labeled protein as a probe. Library screening method (Reference 3; Lowen
stein, EJ et al., Cell 70 (1992) 431-442), a method for screening a cDNA library using an oligonucleotide corresponding to a consensus amino acid sequence of a known SH2 domain as a probe (Reference 4; Matuoka). , K. et al., Proc. Acad. Sci. USA 89 (19
92) 9015-9019), cloned independently and later found to be the same gene. Thus, "functional cl" focusing on the function and structure of protein
Many genes have been conventionally isolated by the "oning method." However, isolating a gene for a novel protein whose function or structure is completely unknown by this method usually involves
Have difficulty.

[0003]

The present inventors have focused on the important role that DNA methylation plays in mammalian development in studies aimed at isolating novel proteins involved in brain-specific signaling. However, it is highly probable that loci on the genome where methylation changes at the developmental stage contain genes encoding proteins with specific functions, especially loci where methylation changes specifically in the brain. He came to the idea that it contains genes encoding brain-specific proteins, including signal transducers. That is, DNA methylation is found in C when aligned with CG, and it is well known that it is essential for mammalian development (Literature 5; Li,
E. et al., Cell 69 (1992) 915-926). In particular, methylation in a specific region rich in CG called a CpG island is often considered to be involved in controlling the expression of a specific gene (Reference 6; Jost, JP and Saluz, HP).
Ed.DNA Methylation: Molecular Biology and Biologic
al Significance (1993) Birkhauser), Changes in methylation can have important implications. Therefore, at the stage of development, loci at which methylation changes brain-specifically in CpG islands or CpG-rich regions on the genome include:
It is likely that genes encoding brain-specific proteins (including those involved in signal transduction) are included.

[0004] Therefore, from such a viewpoint, the present inventors first detected, by RLGS-M, a locus in the mouse brain genome where methylation changes during development.
And the RLGS-M method, a genomic DNA e.g. Not I
After treatment with a methylation-sensitive restriction enzyme such as described above, labeling is performed and high-performance two-dimensional electrophoresis is performed. In this case, an unmethylated Not I site is cleaved and labeled (restriction landmark), so that a spot appears. However, if methylated, the enzyme cannot act and is not labeled, so a spot appears. No (Ref. 8; Hayas
hizaki, Y. et al., Electrophoresis 14 (1993) 251-25
8). The present inventors have efficiently found and reported a locus at which methylation changes during brain development from many loci by this method (Reference 9; Kawai, J. et al., Nucleic Acids Res. 21 (19)
93) 5604-5608). Furthermore, since a restriction enzyme such as Not I cuts a CpG-rich region such as a CpG island (GC / GGCCGC), the locus corresponding to the emerging spot corresponds to or near a specific gene. (Ref. 11; Watanabe, S. et al., Electroph
oresis 16 (1995) 218-226). Thus, DNA fragments were recovered from spots corresponding to loci in which methylation had changed (Reference 12; Suzuki, H. et al., DNA Res. 1 (1994) 1
75-180), and by cloning the gene using it as a probe, the present invention was completed.

[0005] That is, the present invention provides a protein having a cysteine-rich domain and an SH3 domain. Since the protein of the present invention has a novel SH3 domain and a novel cysteine-rich domain, it is expected to be involved in brain-specific signal transduction. The present invention also provides a DNA encoding a protein related to the above novel brain-specific signal transduction.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the protein of the present invention, DNA encoding the same, and a method for producing the same will be described in detail. In the present specification, the novel protein of the present invention is collectively referred to as `` Stac '', particularly mouse-derived Stac is mouse Stac or mStac, and human-derived Stac is human Stac or hSt.
Called Stac. Further, DNA encoding the Stac is
It is referred to as StacDNA, mStac DNA, hStac DNA, etc., but may be simply described as Stac. For the purposes of the present invention, DNA encoding Stac is a synthetic DNA (eg, cD
NA) or natural DNA (genomic DNA), including all of them.

[0007] As described above, Stac of the present invention is disclosed in Reference 8 (Ha
According to the method described in Yashizaki, S et al., supra), genomic DNA derived from mouse brain at various stages of development is treated with a methylation-specific restriction enzyme Not I, and then subjected to RLGS-M for CpG island or CpG-rich. As a result of searching for loci whose methylation changes in the region, one of the positive spots was found.
And is encoded by a gene associated with the corresponding locus. Specifically, as a result of examination, out of about 2600 loci, 44 showed a change in methylation (Reference 9 (Kawai, J. et al.).
Et al., Supra), in particular, see FIGS. Using a DNA fragment obtained from the gel having each of these spots as a probe, c
Screening of the DNA library revealed that the gene corresponding to spot # 91 was the gene encoding the desired protein mStac.

That is, D near the spot # 91 DNA
Using a NA fragment (about 800 bp) as a probe, a mouse brain cDNA library was hybridized, and a positive clone (pMC
SP91; insert size 2396 bp) was obtained.
Furthermore, four clones (pMCSP91R1 to R4) containing the 5 'end of the gene were obtained by RT-PCR (reverse transcription polymerase chain reaction), and were related to spot # 91 based on the nucleotide sequences of these clones. The nucleotide sequence of the full-length cDNA encoding mouse Stac was determined (see FIGS. 1 and 2). In FIG. 2, the underlined part is po
Represents the lyA signal. The nucleotide sequence and the deduced amino acid sequence of mStac DNA are shown in SEQ ID NO: 1.

[0009] Furthermore, c encoding the above-mentioned mouse Stac
Using the DNA as a probe, a human brain cDNA library (Clontech) was screened to obtain cDNA encoding human Stac, and the nucleotide sequence was determined. The nucleotide sequence of the hStac DNA thus obtained and the deduced amino acid sequence are shown in SEQ ID NO: 2. As described later, the Stac of the present invention has a structure (SH3 domain and cysteine-rich domain) characteristic of a brain-specific signal transmitter, and is confirmed from Northern blot results to be specifically expressed in the brain. Was done. Therefore, the novel protein Stac of the present invention is useful for studying the mechanism of brain-specific signal transmission, and ultimately is useful for treatment and prevention of brain abnormalities related to brain signal transmission. In addition, an anti-Stac monoclonal antibody can be prepared and used for research on brain-specific signal transduction, diagnosis of diseases, and the like.

[0010] As will be readily appreciated by those skilled in the art, the DNA of the present invention encodes by modifying the sequence of the DNA by deletion, insertion or substitution of one or more nucleotides in a known manner. The amino acids that make up Stac can be modified to produce improved Stac from humans or mice, or from other mammals, that have desirable or equivalent biological properties to native Stac. . Therefore, proteins and DNAs modified by such a method are also included in the scope of the present invention. Therefore, the protein of the present invention includes a protein having the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence in which one or several amino acids are deleted, substituted or added to the sequence.

That is, the present invention also relates to a cysteine-rich domain comprising the amino acid sequence of amino acid residues 108 to 159 of SEQ ID NO: 2 or the substitution, deletion, insertion or addition of one or several amino acid residues in the sequence. Having at least one mutant sequence thereof selected from among the above, and wherein the SH3 domain has the amino acid residue of amino acid residues 292 to 339 of SEQ ID NO: 2 or the substitution of one or several amino acid residues in the sequence;
It is intended to provide a protein having a mutant sequence thereof having at least one selected from deletion, insertion and addition. Further, the present invention relates to amino acid residues 1 to 2 of SEQ ID NO: 2.
It is intended to provide a protein comprising the amino acid sequence of No. 402 or a mutant sequence thereof having at least one selected from substitution, deletion, insertion and addition of one or several amino acid residues in the sequence. The present invention also provides a polynucleotide molecule (DNA) encoding the novel protein of the present invention. In one embodiment, the polynucleotide molecule of the present invention comprises the nucleotide sequence from C at position 361 to C at position 516 of SEQ ID NO: 2. In another aspect, the polynucleotide molecule of the present invention comprises a G to 1056 G from position 913 of SEQ ID NO: 2.
Includes the base sequence up to position T. Further, the present invention provides a method for producing Stac. To produce such a protein, the Stac DNA of the present invention is inserted into an appropriate vector, and a host cell is transformed with the obtained recombinant vector.
Culturing the transformant and recovering the product from the medium or the cells. Therefore, the present invention provides a recombinant vector containing the polynucleotide molecule of the present invention, and a transformant containing the recombinant vector.

For the expression of the DNA encoding Stac of the present invention, an appropriate one can be selected from expression vector-host systems known to those skilled in the art. Next, the Stac DN of the present invention is placed downstream of the promoter of the selected expression vector.
A can be produced by inserting A into an appropriate host, followed by culturing. As a host for transformation with the expression vector carrying the Stac DNA of the present invention, prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as S. cerevisiae, and higher animal cells and mammalian cells can be used. Can be

Expression vectors suitable for using prokaryotic cells, particularly E. coli, as hosts are known.
Some use a lac promoter or a tac promoter. In order to express Stac DNA in an E. coli host, this DNA must be linked to an E. coli gene expression system in advance. Many books have already been published on expression systems for E. coli [eg, Maniatis et al., (198
2), Moleular Cloning: A Laboratory Manual, Cold Sprin
g Harbor Laboratory].

Examples of a promoter for expression in yeast include the ADH1 promoter and GAL1 promoter, and examples of a promoter for expression in mammalian cells include the SRα promoter and CMV promoter. Transformation of a host cell with an expression vector is known and described in Molecular Cloning: A LAB.
OLATORY MANUAL, Cold Spring Harbor
It can be performed by the method described in Laboratory Press. For example, in the case of a prokaryotic host, a competent cell preparation method, an electroporation method, in the case of a eukaryotic host, a competent cell preparation method, an electroporation method, in the case of a mammalian cell, a transfection method, It can be performed by a poration method. Next, the obtained transformant is cultured in an appropriate medium. When the produced Stac is present in the culture solution or culture filtrate (supernatant), the culture is filtered or centrifuged. From the culture filtrate, conventional methods commonly used for the purification and isolation of natural or synthetic proteins (eg, dialysis, gel filtration, affinity column chromatography using anti-Stac monoclonal antibody, using an appropriate adsorbent) Column chromatography, high performance liquid chromatography, etc.) can be used to purify Stac. When the produced Stac is present in the periplasm and cytoplasm of the cultured transformant, the cells are collected by filtration or centrifugation, and the cell walls and / or
Alternatively, the cell membrane is broken by, for example, ultrasonic treatment and / or lysozyme treatment to obtain debris (crushed cells). The debris is dissolved in a suitable aqueous solution (eg, Tris-HCl buffer). From this solution, Stac
Can be purified.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. The animals used in this example and their sources are as follows. C3H / HeN mouse (Charles River Japan Inc.); BALB / c mouse (SLC Inc.) Various restriction enzymes and kits used in the examples were used in a usual manner according to the instructions of their suppliers. Example 1 Cloning of cDNA Encoding a Mouse Brain-Specific Signaling Substance (1) Detection of a Mouse Brain-Derived Loci That Shows Methylation Changes at Developmental Stages by RLGS-M Various stages of development (embryogen 9 .5 days, 13.5 days, 16.5
Genomic DNA on day 1, 1 day after birth, 10 days, 8 weeks)
Was prepared from the brains of C3H / HeN mice as described in the literature [Hatada, I. et al., Proc. Natl. Acad. Sci. USA 88 (19
91) 9523-9527) and Reference 9 (Kawai, J. et al., Supra)].
Next, the obtained genomic DNA was subjected to methylation-sensitive endonuclease Not I (Takara; 10 units per μg of DNA).
s) at 37 ° C. for 60 minutes, and [α- ³² P] d as usual.
It was end-labeled with GTP and [α- ³² P] dCTP. Labeled DNA the B am HI (Takara, 10u per DNA1μg
nits) at 37 ° C. for 60 minutes, and electrophoresis in the first dimension by 1% agarose gel electrophoresis.
al agarose gel electrophoresis) (4.5 V / cm). After completely digesting the DNA in the agarose gel with HinfI in the system, the agarose gel was ligated to a 6% polyacrylamide gel and subjected to second-dimensional electrophoresis (secondary electrophoresis).
dimensionnal agarose gel electrophoresis) (11
V / cm). Next, the polyacrylamide gel was dried and subjected to autoradiography. By performing the above, the Not I site that is not methylated is cleaved and labeled, and becomes a restriction landmark.
A spot appears, but if methylated, the enzyme does not act and is not labeled, so no spot appears. Thus, for example, N- methylated at the first stage of development
If any of the o t I sites is demethylated (changes in methylation) during development, spots will appear at those sites. Such spots were screened for genomic DNA prepared from mouse brains at each developmental stage, as described above. About 2,600 loci were examined, and 44 loci with altered methylation were found (Reference 9 (Kawa
i, J. et al., supra)). Of the 44 spots corresponding to loci whose methylation changes during development obtained in (1) above, the brain-specific gene Stac was detected from spot # 91 by the following method. As the spot # 91, a genome from an 8 week old animal was used.

(2) Cloning of Gene (cDNA) Corresponding to Spot # 91 Poly (A) ⁺ RNA derived from the brain of C3H / HeN mouse was purified by the following method. That is, Chomezynski and Sacchi (Ana
l Total RNA was extracted from adult mouse brain by the method of Biochem. 162 (1987) 156-159, and oligotex-dT30 (Roche)
Was used to purify Poly (A) ⁺ RNA.

A cDNA was synthesized from the obtained Poly (A) ⁺ RNA and cloned into a plasmid pSPORT1 using a Superscript Plasmid System (Gibco BRL). The cDNA sublibrary comprising each sublibrary per 10 ⁵ independent cDNA clones were prepared in the above 1). On the other hand, a predetermined DNA fragment having a Not I site at the end was recovered from spot # 91 (Reference 12 (Suzuki, H .;
Using the phage vector EMBL3 (Stratagene) to screen a mouse genomic DNA library prepared using the phage vector EMBL3 (Stratagene), about 800 bp.
Was obtained. Next, the newly obtained genome DN
Northern fragment was performed on mouse brain poly (A) ⁺ RNA using fragment A as a probe.
b and hybridized with RNA (Reference
11 (Watanabe, S. et al., Supra)). So, about 800b
When the above mouse brain cDNA library was screened with the p probe, a 239 bp cDNA (approximately 650 bp matched with the probe) was obtained. However, since the 5 'side is a little short for full-length cDNA, 5'-AmpliFINDER based on RT-PCR was used.
With the RACE Kit (Clontech), a clone containing the 5 'end was also obtained.

For the RT-PCR, the Poly obtained by the above method was used.
(A) ⁺ RNA and the following D
5'-AmpliFINDER RACE Kit (Clonte
ch-) according to the protocol. Primer 218 (corresponding to No. 489-364) 5'-GTGGTTGCAG ACATCACAGA AGGTG-3 '(SEQ ID NO: 3) Primer 108 (corresponding to No. 269-245) 5'-CTCCGCAAGC TCTTGGTCTT GAAAG -3' (SEQ ID NO: 4) Thus, four clones (pMCSP91R1)
~ R4), and the sequence is AutoRead DNA sequencing ki
t was determined by ALF DNA sequencer (Pharmacia). Together with the sequence of the clone pMCSP91 obtained in 1) above, the nucleotide sequence of a full-length cDNA encoding a mouse brain-specific signal transmitter (mStac) was determined (FIG. 1).
And 2). The nucleotide sequence and the deduced amino acid sequence of this mStac cDNA are shown in SEQ ID NO: 1.

Example 2 Cloning of cDNA Encoding a Human Brain-Specific Signaling Substance A cDNA library prepared from Poly (A) ⁺ RNA (Clontech) derived from human fetal brain using the mStac cDNA cloned in Example 1 as a probe. Rally (Ref. 17; Orita, S. et al., B
iocchem.Biophys.Res.Commun. 206 (1995) 439-448)
From this, a human Stac clone (hStac cDNA) homologous to mouse Stac cDNA was isolated by cross-hybridization using mouse Stac cDNA (open reading frame, ORF) as a probe, and the entire nucleotide sequence was determined. The nucleotide sequence and the deduced amino acid sequence of this hStac cDNA are shown in SEQ ID NO: 2.

Example 3 Expression of Stac cDNA by NIH3T3 Cells (1) Cell Culture The Stac cDNA obtained in Example 1 was expressed by NIH3T3 cells (ATCC, No. CRL1658), a cell available from the American Type Culture Collection. I let it. NIH
3T3 cells and transformants (transfectants) were prepared in DMEM (Nis) supplemented with 10% fetal bovine serum (Integen).
sui2). Cultures of these cell lines are 5% CO ₂
The test was performed under air containing.

(2) Construction of Expression Vector The full-length Stac cDNA obtained in Example 1 was converted to the expression vector pM.
An expression vector pMIK-mStac was constructed by inserting IK-neo (provided by Professor Kazuo Maruyama, Tokyo Medical and Dental University) downstream of the SRα promoter (for forced expression).

(3) Transfection Using the construct obtained in the above (2), LipofectAMINE ^™ (Gibc
oBRL) was used to transfect the NIH3T3 cells cultured in (1), and the obtained transfectants were cultured under the above conditions. The culture was then transferred to G418 (3
(Μg / ml), a clone of a stable transformant was selected, and mS
The expression of tac was confirmed.

Test Example 1 Structural Analysis of mStac and hStac mStac DNA and hStac DN prepared in Examples 1 and 2
A and the amino acid sequences of the proteins encoded by these DNAs were compared and examined. The homology was 85% for the nucleotide sequence encoding the amino acid sequence and 83% for the amino acid sequence. Each of these sequences is novel. Examination of the hydrophobicity as a structural feature suggests that Stac has no signal peptide or transmembrane portion, and therefore is neither a secretory protein nor a membrane protein. As a result of the homology search, it was found that there were two domains, a cysteine-rich domain and an SH3 domain, which are commonly involved in signal transduction of mStac and hStac (see FIG. 3). The amino acid sequences of these two domains were both novel. The position on the Stac of the cysteine-rich domain is, in the case of mStac, amino acids 109 to 160 of SEQ ID NO: 1, hStac
Corresponds to amino acids 108 to 159 of SEQ ID NO: 2. In addition, in the case of mStac, the position of the SH3 domain is amino acid numbers 293 to 340 of SEQ ID NO: 1, hStac
Corresponds to amino acid numbers 292 to 339 of SEQ ID NO: 2.

Test Example 2 Detection of Stac Protein in Brain Extract by Western Blotting (1) Preparation of Anti-Stac Antibody The N-terminal part (amino acid No. 1-116) of mouse Stac was used as a GST fusion protein as follows. Created.
That, Na e I of mStac shown in SEQ ID NO: 1 (101) - Mse I ( 455)
The DNA fragment was blunt-ended and inserted into the SmaI site of the plasmid pGEX-4T-1 (Pharmacia) for preparing a GST fusion protein.
Using this plasmid, the fusion protein was expressed in Escherichia coli by induction with 0.1 mM IPTG. Next, the fusion protein was
Rabbits were immunized by affinity purification using the GST Purification Module (Pharmacia). First, GS
Antibodies to GST were absorbed through a T column and then affinity purified using a fusion protein column. GS
The T column and its fusion protein column were prepared by cross-linking GST or its fusion protein to Affi-Gel10 or Affi-Gel15 (BioRad).

(2) Western blotting Analysis was performed using NIH3T3 cells transformed with the mStac expression vector described in Example 3 above and a brain extract of an adult mouse (C3H / HeN). NIH3T3 cells into which mStac cDNA had not been introduced were used as controls. Western blots were performed at room temperature. The protein in the Lamuli sample buffer was analyzed by SDS-PAGE (MULTI GEL 10/20, Daiich
and transferred electrically onto a PVDF membrane. Blott membrane
o / Tween (Literature 10; Kawai, J. et al., Mol.Cell.Biol. 14
(1994) 7421-7427).
The cells were incubated with the rabbit anti-Stac serum prepared in (1) (1: 500 dilution) at 25 ° C. for 1 hour. PB
After washing with S / 0.05% Tween 20 for 30 minutes, the membrane was again blocked for 30 minutes. The membrane was then incubated with HRP-conjugated anti-rabbit IgG antiserum (1: 2500 dilution; BioRad) for 1 hour before PBS / 0.05% Tween
Washed at 20 for 1-2 hours. Detection of the signal was performed using the ECL system (Amersham). FIG. 4 shows the results. As is apparent from FIG. 4, a protein having a molecular weight of 47 kD deduced from the amino acid sequence was detected in the brain extract, and this protein was identified as NIH3T transformed with the mStac expression vector.
The proteins from the three cells were identical in size and reactivity with the antibody, and were confirmed to be Stac proteins. Thus, it was confirmed that Stac of the present invention is a protein present in the brain.

Test Example 3 Brain specific expression of Stac In various organs by RNA blotting analysis
Stac distribution was investigated. As a probe for total RNA blotting analysis, mouse Stac c
DNA corresponding to nucleotide residues 122-1169 of DNA
The fragment was used. Poly (A) ⁺ RN prepared from each mouse organ
Commercially available nylon membrane (Multiple Tissue
Northern Blots: Clontech) was used. After hybridization, the nylon membrane is subjected to G3PDH as usual.
After rehybridization using a probe (Clontech), the quality and quantity of Poly (A) ⁺ RNA
The signal was checked against the DH gene mStacRNA.
FIG. 5 shows the results. FIG. 5 shows that Stac is specifically expressed in the brain.

Test Example 4 In Situ Hybridization Using Cryostat from Adult Mouse (BALB / c) Brain
A 15 μm thick frozen section is prepared and fixed with 4% paraformaldehyde. RNA probes for in situ hybridization were prepared from mStac cDNA (base sequence numbers 1168 to 2471) using digoxigenin labeling kit (SP6 or T7 RNA polymerase and digoxigenin-16-
UTP included; made using Boehringer Mannheim GmbH). The obtained digoxigenin-labeled Stac RNA probe (400
ng / ml) was reacted with the brain slice at 50 ° C for 16 hours in a predetermined hybridization buffer (Amersham). After washing twice with 0.1xSSC, the signal was measured by alkaline phosphatase-conjugated anti-
Digoxigenin antibody (Fab fragment; Boehringer Ma
nnheim GmbH). For other conditions, reference was made to the report by Nakayama et al. (Nakayama, M. et al., J. Neuros
ci., 15 (1995) 5238-5248). FIG. 6 shows the results of detection of Stac mRNA in mouse brain by in situ hybridization. In the figure, A to F are the following parts. A: cerebral cortex (Cx) and hippocampus (Hp) B: cerebellum C: medullary olive nucleus D: hippocampus (high magnification), CA and DG represent Ammon's angle and dentate gyrus, respectively. E: cerebellum (high magnification), P, Gr, M represent Purkinje cell layer, granule cell layer and molecular layer, respectively. F: Olive nucleus (high magnification) Scale bars are 2 mm for A, B and C, D, E and F
Represents 400 μm, 250 μm, and 100 μm, respectively. From the figure, Stac expression was clearly observed in brain nerve cells. In particular, cerebral cortex (Figure A), hippocampus Ammon's horn CA1
Strong expression is seen in the region as well as in the dentate gyrus (FIG. D), Purkinje cells in the cerebellum (FIGS. B and E), and the olive nucleus in the medulla oblongata (FIGS. C and F).

[0028]

The protein of the present invention has a cysteine-rich domain and an SH3 domain. The cysteine-rich domain is a domain commonly present in various protein kinases C involved in cell signal transduction (Experimental Medicine vol. 11, No. 15 (extra number) 1993). On the other hand, the SH3 domain is also a tyrosine kinase signal transduction. It is thought to be a domain involved in the system (Experimental Medicine vol.12, No.14 (1
0) 1994). Therefore, it is considered that the protein of the present invention having both of these domains plays an important role in signal transduction in nerve cells when it is specifically expressed in brain nerve cells. Also, various SH3 domains have been found to bind to the proline-rich domain of dynamin I (I. Gout et al., Cell vol. 75,
25-36, 1993). Dynamin I is a protein involved in endocytosis in synaptic vesicles, which is important for neural activity (Experimental Medicine vol.13, No.15 (October) 1995), and is also involved in neurite outgrowth. (J. Biol. Chem. Vol. 269, No. 51, 32411-32417, 19
94). According to these findings, the protein of the present invention acts on dynamin I via the SH3 domain, and may play an important role in vesicle transport and neurite outgrowth in nerve cells. Furthermore, from the above-mentioned action of the protein of the present invention,
Substances that activate the protein of the present invention may be involved in maintenance or activation of neural activity, and therefore, screening such a substance using the protein of the present invention is significant.

[0029]

[Sequence list]

SEQ ID NO: 1 Sequence length: 2517 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin: Organism: Mouse Tissue type: Brain Sequence GCC GCC GCC TGG CGC GAT GGA CAT CCC
GGG GAC CCG GCG GCA CTG AGC 48 GAT CAA GCA CGA GCC TGC GGT TCC CCC
AGA AGC CCA ACA CAG TTC CCG 96 CGG CCG GCC ACG ATG ATT CCT CCA AGT
GGC GCC CGC GAG GAC AGC GGA 144 Met Ile Pro Pro Ser
Gly Ala Arg Glu Asp Ser Gly 12 15
10 GAC GGA CTG ACC GGG GAG GCA ACG GGC
ACA GAG CAG CCG CCC TCT CCT 192 Asp Gly Leu Thr Gly Glu Ala Thr Gly
Thr Glu Gln Pro Pro Ser Pro Pro 28 15 20
25 GCG TCC ACC AGC AGC CTG GAA TCC AAG
CTC CAA AAA CTA AAA CGG TCA 240 Ala Ser Thr Ser Ser Leu Glu Ser Lys
Leu Gln Lys Leu Lys Arg Ser 44 30 35
40 CTT TCT TTC AAG ACC AAG AGC TTG CGG
AGC AAA AGT GCT GAC AAC TTC 288 Leu Ser Phe Lys Thr Lys Ser Leu Arg
Ser Lys Ser Ala Asp Asn Phe 60 45 50
55 60 TTC CCA AGA ACC AAC AGT GAT GTG AAA
CCG CAA GCA GAC CTG CTG GCC 336 Phe Pro Arg Thr Asn Ser Asp Val Lys
Pro Gln Ala Asp Leu Leu Ala 76 65
70 75 AAG GCC AGC CCA GGC CCC AGC CCA ATA
GCC ATC CCT GGA AGC CCA GCG 384 Lys Ala Ser Pro Gly Pro Ser Pro Ile
Ala Ile Pro Gly Ser Pro Ala 92 80 85
90 TCC ATG CCC ACC AAG GCT GGC CTG CAC
CCC GGC AGC AAC AGC AAG CTC 432 Ser Met Pro Thr Lys Ala Gly Leu His
Pro Gly Ser Asn Ser Lys Leu 108 95 100
105 CAC GCC TTT CAG GAG CAT GTC TTT AAG
AAG CCC ACC TTC TGT GAT GTC 480 His Ala Phe Gln Glu His Val Val He Lys
Lys Pro Thr Phe Cys Asp Val 124 110 115
120 TGC AAC CAC ATG ATC GTG GGA ACA CAT
GCT AAG CAC GGA CTG CGC TGC 528 Cys Asn His Met Ile Val Gly Thr Thr His
Ala Lys His Gly Leu Arg Cys 140 125 130
135 140 GGA GCC TGT AAG ATG AGC ATC ATC CAC CAC
AAG TGT GCA GAT GGC CTG GCG 576 Gly Ala Cys Lys Met Ser Ile His His
Lys Cys Ala Asp Gly Leu Ala 156 145
150 155 CCC CAG CGG TGC ATG GGC AAG TTG CCA
AAG GGA TTT CGG CGT TAC TAC 624 Pro Gln Arg Cys Met Gly Lys Leu Pro
Lys Gly Phe Arg Arg Tyr Tyr 172 160 165
170 AGC TCC CCC TTG CTC ATT CAC GAA CAG
TTT GGA TGC ATT AAA GAA GTT 672 Ser Ser Pro Leu Leu Ile His Glu Gln
Phe Gly Cys Ile Lys Glu Val 188 175 175 180
185 ATG CCC ATT GCG TGT GGC AAT AAA GTG
GAC CCC GTG TAT GAG GCC CTC 720 Met Pro Ile Ala Cys Gly Asn Lys Val
Asp Pro Val Tyr Glu Ala Leu 204 190 195
200 CGG TTC GGC ACG TCC CTG GCC CAG AGA
ACG AAG AAG GGC GGC TCA GGC 768 Arg Phe Gly Thr Ser Leu Ala Gln Arg
Thr Lys Lys Gly Gly Ser Gly 220 205 210
215 220 AGT GGT TCT GAC TCA CCG CCC AGA ACC
TCG ACT TCA GAG CTT GTA GAC 816 Ser Gly Ser Asp Ser Pro Pro Arg Thr
Ser Thr Ser Glu Leu Val Asp 236 225
230 235 GTC CCT GAA GAG GCC GAT GGG CCA GGA
GAT GGA TCT GAC ATG AGG ACA 864 Val Pro Glu Glu Ala Asp Gly Pro Gly
Asp Gly Ser Asp Met Arg Thr 252 240 245
250 CGG AGC AAC AGT GTG TTT ACA TAT CCA
GAA AAC GGT ATG GAT GAC TTC 912 Arg Ser Asn Ser Val Phe Thr Tyr Pro
Glu Asn Gly Met Asp Asp Phe 268 255 260
265 AGA GAT CAA ATG AAG ACC ACA AAC CAC
CAG GGA CCT CTT TCC AAA GAC 960 Arg Asp Gln Met Lys Thr Thr Asn His
Gln Gly Pro Leu Ser Lys Asp 284 270 275
280 CCA TTA CAG ATG AAC ACC TAC GTT GCC
TTG TAC AGA TTT ATA CCA CAG 1008 Pro Leu Gln Met Asn Thr Tyr Val Ala
Leu Tyr Arg Phe Ile Pro Gln 300 285 290
295 300 GAG AAT GAA GAC TTG GAA ATG AGG CCA
GGA GAC ATG ATC ACT CTC CTA 1056 Glu Asn Glu Asp Leu Glu Met Arg Pro
Gly Asp Met Ile Thr Leu Leu 316 305
310 315 GAA GAC TCC AAT GAA GAC TGG TGG AAA
GGA AAG ATT CAG GAC AGG GTT 1104 Glu Asp Ser Asn Glu Asp Trp Trp Lys
Gly Lys Ile Gln Asp Arg Val 332 320 325
330 GGT TTC TTT CCA GCC AAC TTT GTT CAG
AGA GTA GAA GAG CAT GAG AAG 1152 Gly Phe Phe Pro Ala Asn Phe Val Gln
Arg Val Glu Glu His Glu Lys 348 335 340
345 ATT TAT AGG TGT GTT CGA ACC TTC ATT
GGC TGC AAG GAC CAG GGG CAG 1200 Ile Tyr Arg Cys Val Arg Thr Phe Ile
Gly Cys Lys Asp Gln Gly Gln 364 350 355
360 ATA ACA CTG AAA GAG AAC CAG ATA TGC
GTG ACC TCT GAG GAA GAA CAG 1248 Ile Thr Leu Lys Glu Asn Gln Ile Cys
Val Thr Ser Glu Glu Glu Gln 380 365 370
375 380 GAC GGC TTC ATC AGA GTC CTC AGT GGG
AAG AAG AGA GGC CTC GTC CCA 1296 Asp Gly Phe Ile Arg Val Leu Ser Gly
Lys Lys Arg Gly Leu Val Pro 396 385
390 395 CTG GAT GTA CTG GTA GAC GTG TGA CTG
TAG CAG TCC CTT CCC GAG CAG 1344 Leu Asp Val Leu Val Asp Val
403 400 403 GAG GCC AGC GCT GCT GAT CTC TCT GTG
TCC ACC CAG AGG AGC AGC TGC 1392 ACG GAT GGG TGC CCA GAA AAC AGG GAG
ACT TGA AGA GCT GAG ACT GTG 1440 CTA GGA ATC TGG AAG CAG GCC CAC TGC
ACA GCA CAT CAA AGC GTC CTG 1488 AGA GCA GCA TCC CAG CGC TGA GGG GAG
CCG GGG GCA GTA AGT GGG CTG 1536 TAG CTA AGG CTG CCC AGG ACT CAG CTC
CCT TCC TGC CCA CCT GTG CAA 1584 GGC TGG AGT CTG CTC CAG GTC ATG GTG
TGG TCC TAG TGA GAA GTT TGG 1632 AGC TGC CGA GTA GCC AAG TTT CTC TGA
TTC TGG ATA ACT GTT CCC AGG 1680 GCT GGC CAA TCA GCC TCA GCA TGA TGA TTC
CTG CAG AGA ATC AAG AAG GTC 1728 AAC CTT CCG GCT CCC AAC ACC CCA GGG
TTC ACT CCA GCT CCT GCT CAG 1776 TTT GTG TCT CAG TTT GGC TGG CAG GAT
TGT TCT TTC TTT CTC TTC AGC 1824 AGC ACA TCC TAG GCA GGG CCG AAC CAG
CAT GGT ACC AAG TGC AAA GAT 1872 CCG ACT TAC CTA CAG ATC GCG AAC GAG
GGC GTC CTT GCT ATT CCA GTG 1920 GCT CCG AAG TGG CAG GGC AAG GTC CAA
GAC ACA GAG TCC AGC AGC AGC 1968 AAG GAG AAG CGG GAG TGG GGA CAA GGA
AGG AGC CCT TAT GTA TTG GGA 2016 TCG GAC AGC CCT TTC AGT TGG GGT GGG
TTT TTT TCC CAA AGC AGG TGA 2064 GGC TGG GAA TGG GAA GTA GGG AAG AGT
CTC CAT TTT CAT TCC ATC CCC 2112 TCT TTA TCA GGA GCA CCA CTT CTT CCT
GTG GAG GAT GAT CCA GGG ACA 2160 GGG GAG CAC CAC TGA GAA AAA CAT CTT
AGA ATA AAT GTA GCC AAC TTA 2208 GAA TCC ATT CTT CTC TAG GGC AAC GTT
TTC AGC TCC AGC AGC TGT GAA 2256 GTG GAA CGG ACC GGG TCA TGC TTC ATC
TCT GTG CAA ACA GAG GAA GCT 2304 GGG GCT TAG CTC ACA CCA CCT GGG GAT
ATT GCT CTC TAC CTG TAC CGA 2352 TGT TTA CAA GAA CAT ACC ACA CTG CGA
TGC CTT CCT CCC AAG CCC CGT 2400 CCC AGC ATT CAG ACC TGC TTC CTA GCA
GTC ACT GAC ATG TTT AAA TGT 2448 GAC TTT GGG ATT ATT GTT GCT TCG ATT
TAT TTT GCC ACT GAG CAA ATA 2496 AAA TCA TTA GAA ACC ATC GGG
2517

SEQ ID NO: 2 Sequence length: 2963 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin: Organism: Human Tissue type: Brain Sequence GTT CCT CCG GGA GCC CAA CAC CGT TCC CGC GCG GCC ACG ATG ATC CCT 48 Met Ile Pro 31 CCG AGC AGC CCC CGC GAG GAC GGC GTG GAC GGG CTG CCC AAG GAG GCG 96 Pro Ser Ser Pro Arg Glu Asp Gly Val Asp Gly Leu Pro Lys Glu Ala 19 5 10 15 GTG GGC GCC GAG CAA CCG CCC TCT CCT GCA TCC ACC AGC AGC CAG GAA 144 Val Gly Ala Glu Gln Pro Pro Ser Pro Ala Ser Thr Ser Ser Gln Glu 35 20 25 30 35 TCC AAG CTC CAG AAA CTA AAA CGA TCA CTT TCT TTC AAG ACC AAG AGT 192 Ser Lys Leu Gln Lys Leu Lys Arg Ser Leu Ser Phe Lys Thr Lys Ser 51 40 45 50 TTA CGG AGC AAA AGT GCT GAC AAC TTC TTC CAG CGA ACC AAC AGC GAA 240 Leu Arg Ser Lys Ser Ala Asp Asn Phe Phe Gln Arg Thr Asn Ser Glu 67 55 60 65 GAC ATG AAA CTG CAA GCA CAC ATG GTG GCT GAG ATC AGC CCC AGC TCC 288 Asp Met Lys Leu Gln Ala His Met Val Ala Glu Ile Ser Pro Ser Ser 83 70 75 80 AGC CCA CTC CCT GCT CCA GGA AGC CTG ACG TCC ACA CCC GCC AGG GCT 336 Ser Pro Leu Pro Ala Pro Gly Ser Leu Thr Ser Thr Pro Ala Arg Ala 99 85 90 95 GGT CTG CAT CCA GGT GGC AAG GCT CAT GCC TTT CAG GAA TAC ATC TTC 384 Gly Leu His Pro Gly Gly Lys Ala His Ala Phe Gln Glu Tyr Ile Phe 115 100 105 110 115 AAG AAG CCC ACT TTC TGT GAT GTC TGC AAC CAC ATG ATA GTG GGA ACA 432 Lys Lys Pro Thr Phe Cys Asp Val Cys Asn His Met Ile Val Gly Thr 131 120 125 130 AAT GCT AAG CAT GGA CTG CGC TGC AAA GCC TGT AAG ATG AGC ATC CAC 480 Asn Ala Lys His Gly Leu Arg Cys Lys Ala Cys Lys Met Ser Ile His 147 135 140 145 CAC AAG TGC ACA GAT GGC CTG GCA CCC CAG CGG TGC ATG GGC AAG CTG 528 His Lys Cys Thr Asp Gly Leu Ala Pro Gln Arg Cys Met Gly Lys Leu 163 150 155 160 CCA AAG GGG TTT CGG CGT TAC TAC AGC TCC CCC TTG CTC ATT CAT GAA 576 Pro Lys Gly Phe Arg Arg Tyr Tyr Ser Ser Pro Leu Leu Ile His Glu 179 165 170 175 CAG TTT GGC TGC ATT AAA GAA GTT ATG CCC ATT GCC TGT GGC AAT AAG 624 Gln Phe Gly Cys Ile Lys Glu Val Met Pro Ile Ala Cys Gly Asn Lys 195 180 185 190 195 GTG GAC CCT GTC TAC GAG ACC CTC CGC TTC GGC ACC TCC CTG GCC CAG 672 Val Asp Pro Val Tyr Glu Thr Leu Arg Phe Gly Thr Ser Leu Ala Gln 211 200 205 210 AGG ACA AAG AAG GGC AGC TCC GGC AGT GGC TCT GAC TCA CCT CAC AGA 720 Arg Thr Lys Lys Gly Ser Ser Gly Ser Gly Ser Asp Ser Pro His Arg 227 215 220 225 ACC TCT ACT TCA GAT CTT GTG GAG GTT CCT GAG GAA GCC AAT GGG CCA 768 Thr Ser Thr Ser Asp Leu Val Glu Val Pro Glu Glu Ala Asn Gly Pro 243 230 235 240 GGA GGC GGG TAT GAC CTA AGG AAA CGC AGC AAC AGC GTG TTT ACA TAT 816 Gly Gly Gly Tyr Asp Leu Arg Lys Arg Ser Asn Ser Val Phe Thr Tyr 259 245 250 255 CCA GAA AAT GGC ACT GAT GAT TTC AGA GAT CCA GCG AAG AAC ATA AAC 864 Pro Glu Asn Gly Thr Asp Asp Phe Arg Asp Pro Ala Lys Asn Ile Asn 275 260 265 270 275 275 CAC CAG GGA TCT CTT TCC AAA GAC CCA TTA CAG ATG AAC ACC TAT GTT 912 His Gln Gly Ser Leu Ser Lys Asp Pro Leu Gln Met Asn Thr Tyr Val 291 280 285 285 290 GCC TTG TAC AAA TTT GTA CCA CAG GAG AAT GAA GAT TTG GAA ATG AGG 960 Ala Leu Tyr Lys Phe Val Pro Gln Glu Asn Glu Asp Leu Glu Met Arg 307 295 300 300 305 CCA GGA GAC ATA ATT ATT ACT CTT TTA GAG GAT TCC AAT GAA GAC TGG TGG 100 Pro Gly Asp Ile Ile Thr Leu Leu Glu Asp Ser Asn Glu Asp Trp Trp 323 310 315 320 AAA GGG AAA ATT CAA GAC AGA ATT GGC TTC TTT CCA GCC AAC TTT GTT 1056 Lys Gly Lys Ile Gln Asp Arg Ile Gly Phe Phe Pro Ala Asn Phe Val 339 325 330 335 CAG AGA CTA CAA CAA AAT GAG AAG ATT TTT AGA TGT GTT AGA ACC TTC 1104 Gln Arg Leu Gln Gln Asn Glu Lys Ile Phe Arg Cys Val Arg Thr Phe 355 340 345 350 355 ATT GGG TGT AAG GAA CAG GGG CAG ATA ACA CTG AAA GAG AAT CAG ATC 1152 Ile Gly Cys Lys Glu Gln Gly Gln Ile Thr Leu Lys Glu Asn Gln Ile 371 360 365 370 TGC GTG AGT TCT GAA GAA GAA CAA GAT GGT TTT ATC AGA GTC CTC AGT 1200 Cys Val Ser Ser Glu Glu Glu Gln Asp Gly Phe Ile Arg Val Leu Ser 387 375 380 385 GGA AAA AAG AAA GGC CTC ATC CCC CTT GAT GTA CTA GAA AAC ATC TGA 1248 Gly Lys Lys Lys Gly Leu Ile Pro Leu Asp Val Leu G lu Asn Ile 402 390 395 400 402 TTG CTG GCT CCT CCT CCG TTT GCA GTA GGC AAG CTC TGC TGC GAT GCC 1296 TCT GCC TCA TCT CAC ACT GCG TCA ACC CAA AGG AGC TGC CGC ACT GAC 1344 CCA GCC CCC CAG GAA ACA GTG AGA CAA GAA TCA AGT ATC TGA GAC TGT 1392 GGA GTA ATA GCC ACA AAA CAG AGG GCC CAC TGC ACA GCA TAT CCA GGC 1440 TGC CAC AGG TGG GGA CGA GGC TGA GAG AGT CAG CAG GCA GAG CCA GAT 1488 GCC ATG CTT GGC AGC AGC AGG ACT ATA AAC CAC AGC TGT CCC CCA 1536 GGA TCC CAC TCC TTT CCT GTC TGT GTG GTG TAA GTT AAC ACA CTG GAG 1584 TGT GCT CCA GTT TGC AGG GTA GCC CAG TGC AAG GTT CAG ATC CAT GTA 1632 GCT AAG TAT CAT TCC AGA CCT ATG TCA CCA GTA CCA ATC AGT 1680 CAG TGT CAT CAC ATT TCA GGC CCC AAG CAA TCT CTG TGC AAA GCA TCA 1728 GAA AGA CCT GCT TCC CAG CCC CCA GCA TTC CAG TGC TCT CCA GGC TTC 1776 CTC TCT TTG TGA TGC TGT CCA GAG TGT CCA GCT TGT TCT TTC TTT 1824 CTC TTC AGT CCT CTG AGT ACA TCT GGT GGT GTG CAT TAG ATG TGA GGG 1872 CTA TGT TGA CAT GGC ATC ACC TCC AAA GAC CTG ACC TGC CTA AAG ACT 1920 GAT GAC AGG CCA TCC TTC CTG CTG TTC TAG GTA CTG GCC TGG GTG ACA 1968 GAG CAG GAC ATG AGA CAT AGA TAC AGT GGG GAG GAG AAG TGG GGA AAG 2016 GTG GAG CAG AGA GTT CTT ACT TAT TGA AGA TTA TAC AGC CCT TTC GTC TAT GAA GTC CCT GCT TGA AGG CAA TGG ACC TGG GGA AGA GAC TAT CAC 2112 AAA AAG TCT CCA TTT TCA TTT TAC ATC CTC TCT ATT GGA GGC AGC ACT 2160 TTT CCC TCA TGC TGT CCT ATA GGA CTC CAC TTT GAA GGT TGT GCC 2208 GTT GCA GGG AAC TAG GAA CAT GGA GGG GAA CCA ACA ACA GCA TCT TAG 2256 AAG AAA TGT AGC CAA ATT GGA GTC CAT TCT TCT TTA GGG CAG TAT ATG 2304 AAA TCC TAG CAG ATG TAA AAT GGA AAA GAA TCC TAA TGC TTCTC CTT 2352 CAG AAA GTA GAG GAA CTA GGG GCC CAA TTA GCA TCA TCT AGG GGA ATC 2400 TCT ATT ACT CTG TAC TTA TAC TAA TGT TTA CAA GAA TGC AAT ATA CTG 2448 TGA TGC CTT CCT ACT CAA GCC TCC TAG CAT TCA ATC TTC CCT ATT 2496 AGT CAT TAA CGT GGT TAA ACT TCA ATT CAC AAT CAC CTT GGA ATC AAT 2544 GTC AGT TTG ATT TAT TTT GTT ACA GAG CAA TAA AAT CAT TAG AAC AAT 2592 GGT TTT TAA AAG ACT TAA GTG GAT GCA T CC TAT GCA TGT AAG CAT TAT 2640 TTC CCA AAC CAA AGC ATC ATT CAT CTC CAT TTA TTT CTT TTG TTC CCG 2688 CTC AGT GTG AAG TTG GGA ACT GAG AGG GGA TGG CTG CTG GTT TCA CAG 2736 AAG TTT GGA TGC CTT ACT CTT TTA AAG CCA GCA TCC AGA ATT TCC 2784 TCC CTC TCT GAT GCC ACA TGC AAA ACC AGG TGT ACG ATG TCA AGA TGG 2832 ATT CTT TGA GAG CCA GGG TAG ATC ATA TGA CTG CCT TTC TGT AAA ATT 2880 GGA TGC CTA GTA CAATC C TTT GCC TCT AAT TCA GTA TTC CAT TTA 2928 ATA AAA ACA ATA CAG TGG CTG GAA AGG AGC ATC AG 2963

SEQ ID NO: 3 Sequence length: 25 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GTGGTTGCAG ACATCACAGA AGGTG 25

SEQ ID NO: 4 Sequence length: 25 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence CTCCGCAAGC TCTTGGTCTT GAAAG 2
5

[Brief description of the drawings]

FIG. 1 is a schematic diagram of cloning of DNA corresponding to spot # 91 in RLGS-M of genomic DNA derived from mouse brain.

FIG. 2 shows the nucleotide sequence of mouse Stac cDNA.

FIG. 3 is a comparison diagram of the amino acid sequences of mStac and hStac.

FIG. 4. NIH3T3 cells transformed with mStac cDNA expression vector, NIH3T not transformed
FIG. 4 is a schematic view of a Western blot of a 3 cell and mouse brain extract using an anti-Stac antibody.

FIG. 5 is a schematic diagram of a Northern blot performed using poly (A) RNA obtained from various organs (heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis) of a mouse.

FIG. 6 is a simulated view of the results of detection of Stac mRNA at various sites in mouse brain by in situ hybridization.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91)

Claims (9)

[Claims] 1. A protein having a cysteine-rich domain and an SH3 domain. 2. The cysteine-rich domain is at least one selected from substitution, deletion, insertion, and addition of one or several amino acid residues in the amino acid sequence of amino acid residues 108 to 159 of SEQ ID NO: 2 or the sequence thereof. The SH3 domain has one or more amino acid residues 292 to 339 of SEQ ID NO: 2 or a substitution, deletion, insertion, or addition of one or several amino acid residues in the sequence. The protein according to claim 1, which has a mutant sequence thereof having at least one selected from the group consisting of: 3. An amino acid sequence of amino acid residues 1 to 402 of SEQ ID NO: 2 or a sequence having at least one selected from substitution, deletion, insertion and addition of one or several amino acid residues in the sequence. The protein according to claim 1, comprising a mutant sequence. A polynucleotide molecule encoding the protein according to any one of claims 1 to 3. 5. The polynucleotide molecule according to claim 4, which comprises a base sequence from C at position 361 to C at position 516 of SEQ ID NO: 2. 6: 1056 from G at position 913 of SEQ ID NO: 2
The polynucleotide molecule according to claim 4, comprising a base sequence up to position T. 7. A recombinant vector containing the polynucleotide molecule according to any one of claims 4 to 6. A transformant comprising the recombinant vector according to claim 7. 9. The method for producing a protein according to claim 1, wherein the transformant according to claim 8 is cultured, and the product is recovered from the medium or the cells.