JP2001333775A - Kinesin superfamily motor protein kif17 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify a new microtubule-binding motor protein and elucidate the mechanism of transportation of a neurotransmitter receptor by the protein. SOLUTION: This protein is any of the following (a) to (c) (a) a protein having a specific amino acid sequence derived from a human, (b) a protein having an amino acid sequence in which one to plural amino acids are deleted, substituted and/or added in the above amino acid sequence and having an activity of binding to an mLin-10 and transporting an N-methyl-D-aspartate(NMDA) receptor 2B(NR2B) and (c) a protein having an amino acid sequence having the homology of >=60% with the above amino acid and further having an activity of binding to the mLi-10 and transporting the NMDA receptor 2B(NR2B).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel neuron-specific microtubule-associated motor protein, a gene encoding the protein, an antibody recognizing the protein, and methods of using them.

[0002]

BACKGROUND OF THE INVENTION In mammalian neurons, neurotransmitter receptors, such as the N-methyl-D-aspartate receptor (NMDAR), are distributed dramatically and accurately to cell dendrites (RSpetralia et al., J. Neurosci. 14, 6102 (1994);
MERubio et al., Neuron 18, 939 (1997); M. Rubio et al., JN
euroscience 19, 5549 (1999); and SJSmith, Science
283, 1860 (1999)). Putative anchor, partition, and signal molecules co-localize with the receptor (M.
D. Ehlers et al., Curr. Opin. Cell Biol. 8, 484-9 (1996); MS
Heng et al., Curr. Opin. in Neurobiol. 6, 602 (1996); and MB.
Kennedy, TrendsNeurosci. 20 264 (1997)), the mechanism of receptor distribution is unknown. Transport of molecules to specific sites in eukaryotic cells is achieved by molecular motors (N.Hi
rokawa, Science 279, 519 (1998); and DLFoletti et al.
Neuron 23, 641 (1999)). In neurons, various microtubule-associated motor proteins have been shown to carry membrane organelles, such as synaptic vesicle precursors and mitochondria, to specific sites in cells, but each motor carries its specific transporter. The mechanism of recognition is still unknown (N. Hirokawa, Sci.
ence 279, 519 (1998); and DLFoletti et al., Neuron 23,
641 (1999)).

[0003]

An object of the present invention is to identify a novel microtubule-associated motor protein and to clarify the mechanism of transport of a neurotransmitter receptor by the protein.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, a novel neuron-specific microtubule-dependent motor molecule, KIF17 (T. Nakagaw) has been developed.
USA et al., Proc. Natl. Acad. Sci. USA 94, 9654 (1997) and identified the molecule as mLin-10 (JSSimske et al., Cell 85, 195 (199)
6); DSBredt, Cell 94, 773 (1998); JPBorg et al., J. Bio.
l. Chem. 273, 31633 (1998); and SMKaech et al., Cell 94,
761 (1998)) (Z. Songyang et al., Science 27
5, 73 (1997)) and binds directly to NMDA receptor 2
It was found to transport large protein complexes containing B (NR2B). This complex was also found to transport vesicles along microtubules in neurons such as hippocampal pyramidal neurons. The present invention has been completed based on these findings.

That is, the present invention provides the following (1) to (11)
Is provided. (1) Any one of the following proteins (a) to (c): (A) a protein having the amino acid sequence of SEQ ID NO: 1; (b) an amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted and / or added, and mLin-10 Binds to NMDA receptor 2
A protein having an activity of transporting B (NR2B); and (c) an NMDA receptor 2B having an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 and binding to mLin-10. A protein having an activity of transporting (NR2B).

(2) A DNA encoding the protein of (1). (3) Any of the following DNAs. (A) a DNA having the base sequence of SEQ ID NO: 3; (b) a base sequence having the base sequence of SEQ ID NO: 3 in which one or more bases have been deleted, substituted and / or added, and mLin-10 DNA encoding a protein having an activity of transporting NMDA receptor 2B (NR2B) by binding to; (c) hybridizing with the DNA of (a) or (b) above under stringent conditions; DNA encoding a protein having an activity of transporting NMDA receptor 2B (NR2B) by binding to -10.

(4) The DNA according to (2) or (3)
A recombinant vector comprising: (5) A transformant having the recombinant DNA according to (4). (6) culturing the transformant according to (5) in a medium,
The method for producing a protein according to (1), comprising producing the protein according to (1) in a culture, and collecting the protein from the culture. (7) An antibody or a fragment thereof that recognizes the protein of (1).

(8) A protein complex comprising at least the protein of (1) and mLin-10. (9) A method for screening for a substance that specifically interacts with the protein according to (1) or using the transformant according to (5). (10) A pharmaceutical comprising the protein according to (1) or the DNA according to (2) or (3) as an active ingredient. (11) The medicament according to (10), which is a medicament for treating and / or preventing a disease associated with abnormal nerve cell function.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. (1) Protein of the present invention and DNA of the present invention The present invention relates to any of the following proteins (a) to (c). (A) a protein having the amino acid sequence of SEQ ID NO: 1 (also referred to herein as KIF17); (b) one or more amino acids are deleted, substituted and / or added in the amino acid sequence of SEQ ID NO: 1 Has an amino acid sequence and binds to mLin-10 to bind NMDA receptor 2
A protein having an activity of transporting B (NR2B); and (c) an NMDA receptor 2B having an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 and binding to mLin-10. A protein having an activity of transporting (NR2B).

In the present specification, "1 to a plurality of amino acids are deleted, substituted and / or added" means, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, More preferably, it means that about 1 to 5 arbitrary number of amino acids have been deleted, substituted and / or added.

As used herein, the term "amino acid sequence having at least 60% homology with the amino acid sequence of SEQ ID NO: 1" means at least 60%, preferably at least 70%, more preferably at least 60% with the amino acid sequence of SEQ ID NO: 1. 80% or more, more preferably 90% or more, particularly preferably 95% or more.
%, Most preferably 98% or more homology.

As used herein, “a protein having an activity of binding to mLin-10 and transporting NMDA receptor 2B (NR2B)” refers to a protein of the present invention binding to mLin-10 to form a complex. It refers to the ability to transport NMDA receptor 2B (NR2B) by moving on microtubules while forming and cooperating with several factors. As an example of the protein of the present invention,
The protein having the amino acid sequence of SEQ ID NO: 1 can be mentioned, but the protein of the present invention is described above as long as it has an activity of binding to mLin-10 and transporting NMDA receptor 2B (NR2B). As described above, even a mutant protein in which one or more amino acids are deleted, substituted and / or added has 60%
A homologous protein having the above homology may be used. SEQ ID NO: 1
Variant proteins and homologous proteins having the following amino acid sequences are all within the scope of the present invention.

When preparing the mutant protein and the homologous protein as described above, first, mutations such as addition, deletion and substitution are introduced into a DNA encoding the amino acid sequence of SEQ ID NO: 1 to produce a mutant gene. To build. As a method for introducing a mutation into DNA, a drug that is a mutagen with the DNA,
Specifically, hydroxylamine, nitrous acid, sulfurous acid, 5 '
-Bromouracil and the like. Further, a chemically synthesized oligonucleotide is annealed to obtain a mutant DNA having a mutation at a desired site.
It is also possible to construct In addition, ultraviolet irradiation method,
Methods such as a site-directed mutagenesis method using the cassette mutagenesis method and the PCR method and a random mutagenesis method can be widely used.

Next, the mutant gene is introduced into an appropriate vector / host system to prepare a transformant. From the transformants, the desired activity (ie, binding to mLin-10,
Those which produce a protein having MDA receptor 2B (NR2B) transport activity are screened. A transformant producing a mutant protein or a homologous protein may be cultured in an appropriate medium, and the target protein may be collected from the resulting culture.
The crude protein obtained from the culture was gel filtered, reverse phase HP
The protein can be further purified using a means used for ordinary protein purification such as LC and ion exchange column purification.

The present invention also relates to a DNA encoding the above-mentioned protein of the present invention. Specific examples of the DNA of the present invention include any of the following DNAs. (A) a DNA having the base sequence of SEQ ID NO: 3; (b) a base sequence having the base sequence of SEQ ID NO: 3 in which one or more bases have been deleted, substituted and / or added, and mLin-10 DNA encoding a protein having an activity of transporting NMDA receptor 2B (NR2B) by binding to; (c) hybridizing with the DNA of (a) or (b) above under stringent conditions; DNA encoding a protein having an activity of transporting NMDA receptor 2B (NR2B) by binding to -10.

As used herein, the phrase "one or more bases have been deleted, substituted and / or added" means, for example, 1
~ 20, preferably 1 ~ 15, more preferably 1 ~ 1
It means that an arbitrary number of 10, more preferably 1 to 5, bases has been deleted, substituted, and / or added.

As used herein, "hybridize under stringent conditions" refers to the use of DNA as a probe and colony hybridization, plaque hybridization, Southern blot hybridization, or the like. The obtained DNA means specifically, using a filter on which colony- or plaque-derived DNA or a fragment of the DNA is immobilized, in the presence of 0.7 to 1.0 M NaCl,
After hybridization at 5 ° C, 0.1-2
SSC solution of about 1 times (the composition of 1 time concentration SSC solution is 150 times
DNA that can be identified by washing the filter using 65 mM of sodium chloride and 15 mM sodium citrate) at 65 ° C. Hybridization is performed using Molecular Cloning: A laboratory Mannua
l, 2 ^nD ED., Cold Spring Harbor Laboratory, Cold Sp
ring Harbor, NY., 1989. Hereinafter, it is abbreviated as "Molecular Cloning Second Edition") and the like.

The DNA which can be hybridized under stringent conditions includes a DNA having a certain degree of homology with the base sequence of the DNA used as the probe.
NA is mentioned, and the homology is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

(2) Production of the protein of the present invention using the DNA of the present invention In order to produce the protein by expressing the above DNA in a host cell, the DNA fragment is placed downstream of the promoter in an expression vector. The inserted recombinant expression vector is introduced into a host cell suitable for the expression vector.

As the host cell, any cell that can express the target gene can be used. For example, bacteria (eg, Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc.), yeasts (Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon sp., Siwaniomyces sp.
CHO cells, etc.) or insect cells (Sf9 cells, Sf21 cells, etc.).

When a bacterium is used as a host, an expression vector which is capable of autonomous replication in the above-mentioned host cell or can be integrated into a chromosome and which contains a promoter at a position where the above-mentioned DNA of interest can be transcribed is used. Used. Examples of expression vectors include, for example, pBTrP2, pBTac
1, pBTac2 (all commercially available from Beiler Ringermanheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pQE
-30 (manufactured by QIAGEN), pKYP10 (JP-A-58-110600), pKYP200
(Agrc. Biol. Chem., 48, 669 (1984)), PLSA1 (Agrc.
lo1. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Ac
ad.Sci. USA, 82, 4306 (1985)], pBluescrlptII SK
+, PBluescriptII SK (-) (Stratagene), pTrS30 (FER
MBP-5407), pTrS32 (FERM BP-5408), pGEX (Pharmacia), pET-3 (Novagen), pTerm2 (US4686191, US49390)
94, US5160735), pSupex, pUB110, pTP5, pC194, pUC18
(Gene, 33, 103 (1985)), pUC19 (Gene, 33, 103 (198
5)), pSTV28 (Takara Shuzo), pSTV29 (Takara Shuzo), pUC1
18 (manufactured by Takara Shuzo Co., Ltd.), pPA1 (JP-A-63-233798), pEG400 (J.
Bacterio 1., 172, 2392 (1990)), pQE-30 (QIAGEN)
And the like. As a method for introducing a recombinant vector into a bacterial host, any method for introducing DNA into the above host cells can be used, for example, a method using calcium ions (Proc. Nat1.Acad.SCl.
USA, 69, 2110 (1972)), the protoplast method (Japanese Unexamined Patent Publication No.
2483942), or Gene, 17, 107 (1982) or Molecular & G
eneral Genetics, 168, 111 (1979).

When yeast is used as a host cell, as an expression vector, for example, YEp13 (ATCC37115), YEp24
(ATCC37051), Ycp5O (ATCC37419), pHS19, pHS15 and the like. Any promoter can be used as long as it can be expressed in yeast, such as a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, a gal1 promoter, a gal10 promoter, a heat shock protein promoter, and MFα1.
Promoters such as a promoter and a CUP1 promoter can be mentioned. As a method for introducing the recombinant vector into yeast, any method can be used, for example, an electroporation method [Methods. Enzymol, 194, 182].
(1990)), spheroblast method (Proc. Natl. Acad. Sc.
i. USA, 75, 1929 (1978)], lithium acetate method [J. Bacteri
ol., 153, 163 (1983)], or Proc.
Sci. USA, 75, 1929 (1978).

When an animal cell is used as a host cell, examples of the expression vector include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 [JP-A-3-22979; Cytotechn.
ology, 3, 133, (1990)), pAS3-3 (Japanese Patent Application Laid-Open No. 2-227075), pCD
M8 (Nature, 329, 840, (1987)), pcDNAI / AmP (Invitrog
en), pREP4 (Invitrogen), pAGE103 (J. Bloche
m., 101, 1307 (1987)], pAGE210, and the like. As a method for introducing a recombinant vector into animal cells,
Any method can be used, for example, an electroporation method (Cytotechnology, 3, 133 (1990)), a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a lipofection method (Proc. Nat1.Acad.Sci., USA, 84, 7413 (198
7)], and the method described in virology, 52, 456 (1973).

When an insect cell is used as a host, for example, baculovirus expression vectors, a laboratory manual (Baculovirus Expre
ssion Vectors, A Laboratory Manua1), Current Protocols in Molecular Biology, Bi
The protein can be expressed by the method described in o / Technology, 6, 47 (1988). That is, after the recombinant gene transfer vector and the baculovirus are co-transfected into insect cells to obtain the recombinant virus in the insect cell culture supernatant, the recombinant virus is further infected into insect cells to express the protein. it can. The gene transfer vector used in the method includes, for example, pVL139
2, pVL1393, pBlueBacIII (both from Invitrogen) and the like.

As the baculovirus, for example, Autographa californica nuclei polyhedrosis virus, which is a virus that infects night insects of the family Rothaceae
(Autographa californica nuclear polyhedrosis viru
s) and the like can be used. As insect cells, Spodop
Tera frugiperda ovary cells Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company,
New York, (1992)], Trichoplusia ni
Ovarian cell High5 (Invitrogen) or the like can be used. For preparing a recombinant virus,
Examples of the method of co-introducing the recombinant gene transfer vector and the baculovirus into insect cells include, for example, a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a lipofection method (Pr
oc. Nat1. Acad. Sci. USA, 84, 7413 (1987)].

In the present invention, the above-described recombinant DNA
Is cultured in a culture medium, and binds to the mLin-10 of the present invention in the culture to produce and accumulate a protein having an activity of transporting NMDA receptor 2B (NR2B). The protein can be collected. The method of culturing the transformant carrying the DNA of the present invention in a medium can be performed according to a usual method used for culturing a host. When the transformant of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the culture medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism. Any of a natural medium and a synthetic medium may be used as long as the medium can efficiently culture the transformant. The cultivation is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7
Days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.

When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG) or the like is trp.
When culturing a microorganism transformed with an expression vector using a promoter, indoleacrylic acid (IAA) or the like may be added to the medium.

To isolate and purify a protein having the activity of transporting NMDA receptor 2B (NR2B) by binding to mLin-10 of the present invention from a culture of the transformant of the present invention, a conventional protein May be used. For example, when the protein of the present invention is expressed in a dissolved state in the cells, after completion of the culture, the cells are recovered by centrifugation and suspended in an aqueous buffer,
The cells are crushed by an ultrasonic crusher, a French press, a Manton-Gaurin homogenizer, a Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a normal protein isolation and purification method, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using a resin such as diethylaminoethyl (DEAE) Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), S-Sepharose FF
Cation exchange chromatography using a resin such as (Pharmacia), Ni-NTA agarose, butyl sepharose, hydrophobic chromatography using a resin such as phenyl sepharose, gel filtration using a molecular sieve,
A purified sample can be obtained using a single method or a combination of methods such as affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing.

When the protein is expressed in an insoluble form in the cells, the cells are similarly recovered, crushed, and centrifuged to obtain a precipitate fraction obtained by a conventional method. After recovering the protein, the insoluble form of the protein is solubilized with a protein denaturant. After diluting the lysate into a solution containing no protein denaturing agent or a solution in which the concentration of the protein denaturing agent is so low that the protein is not denatured, or dialyzed to form the protein into a normal three-dimensional structure, A purified sample can be obtained by the same isolation and purification method.

When the protein of the present invention or its derivative such as a modified sugar thereof is secreted extracellularly, the protein or its derivative such as a sugar chain adduct can be recovered in the culture supernatant. That is, the culture is treated by a method such as centrifugation as described above to obtain a soluble fraction, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.

(3) Antibody of the Present Invention The present invention also relates to an antibody that recognizes the protein of the present invention which has an activity of transporting NMDA receptor 2B (NR2B) by binding to mLin-10. The antibodies of the present invention may be polyclonal or monoclonal. Antibody fragments are also within the scope of the present invention. Examples of antibody fragments include F (ab ') 2 fragment, Fab' fragment and the like.

The production of the antibody of the present invention can be carried out by a conventional method. For example, a polyclonal antibody that recognizes the protein of the present invention can be obtained by immunizing a mammal with the protein of the present invention as an antigen, collecting blood from the mammal, and separating and purifying the antibody from the collected blood. be able to. For example, mammals such as mice, hamsters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep, and cows can be immunized. As a method of immunization, for example, J.ASSOC.OFF.ANAL.CHEM 70 (6) 1025-1027
(1987) and the like, using the usual immunization method,
For example, it can be performed by administering the antigen one or more times.

The antigen is preferably administered two or three times, for example, at an interval of 7 to 30 days, particularly 12 to 16 days. The dose can be set, for example, at about 0.05 to 2 mg of the antigen per dose. The administration route is not particularly limited, and subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be appropriately selected.
It is preferred to administer by intravenous, intraperitoneal or subcutaneous injection. In addition, the antigen is a suitable buffer such as complete Freund's adjuvant, RAS [MPL (Monoph
osphoryl Lipid A) + TDM (Synthetic Trehalose Dicoryno)
mycolate) + CWS (Cell Wall Skeleton) adjuvant system), which can be used by dissolving it in an appropriate buffer containing a commonly used adjuvant such as aluminum hydroxide, but depending on the administration route and conditions, the above adjuvant may be used. May not be used. Here, an adjuvant means a substance that, when administered together with an antigen, nonspecifically enhances an immune response to the antigen.

After the immunized mammal is bred for 0.5 to 4 months, a small amount of serum from the mammal is sampled from the ear vein or the like, and the antibody titer is measured. When the antibody titer increases, the antigen is administered an appropriate number of times according to the situation. For example, a booster immunization is performed using 100 μg to 1000 μg of the antigen. One to two months after the last administration, blood is collected from the immunized mammal by a conventional method, and the blood is subjected to, for example, centrifugation, precipitation using ammonium sulfate or polyethylene glycol, gel filtration chromatography, ion exchange. A polyclonal antibody recognizing the protein of the present invention can be obtained as a polyclonal antiserum by separating and purifying by ordinary methods such as chromatography such as chromatography and affinity chromatography. The serum is, for example, 5
The complement system may be inactivated by treatment at 6 ° C. for 30 minutes.

The globulin type of the monoclonal antibody that recognizes the protein of the present invention is not particularly limited.
G, IgM, IgA, IgE, IgD and the like.
Further, the antibody of the present invention is preferably a humanized antibody or a human antibody. The cell line that produces the monoclonal antibody of the present invention is not particularly limited. For example, it can be obtained as a hybridoma by cell fusion between an antibody-producing cell and a myeloma cell line. The hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.

As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used.
The protein of the present invention or its partial peptide is used as the antigen. Mice, rats, and the like are used as animals to be immunized, and administration of the antigen to these animals is performed according to a conventional method. For example, a suspension or emulsion of an adjuvant, such as complete Freund's adjuvant or incomplete Freund's adjuvant, and the protein of the present invention, which is an antigen, is prepared and administered several times to the vein, subcutaneous, intradermal, intraperitoneal, etc. of an animal. The animal is immunized by doing so. For example, spleen cells are obtained as antibody-producing cells from the immunized animal, and these cells and myeloma cells are isolated by a method known per se (G. Kohler et al., Nat.
ure, 256 495 (1975)) to produce a hybridoma.

Examples of myeloma cell lines used for cell fusion include P3X63Ag8 and P3U1 in mice.
And Sp2 / 0 strains. When performing cell fusion, a fusion promoting agent such as polyethylene glycol or Sendai virus is used. For selection of hybridomas after cell fusion, hypoxanthine-aminopterin-thymidine (HAT) medium can be used according to a conventional method. The hybridoma obtained by cell fusion is cloned by a limiting dilution method or the like. Furthermore, by performing screening by an enzyme immunoassay using the protein of the present invention, a cell line that produces a monoclonal antibody that specifically recognizes the protein of the present invention can be obtained.

To produce the desired monoclonal antibody from the thus obtained hybridoma, the hybridoma is cultured by a usual cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites. do it. Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used in appropriate combination.

Examples of the method for immunoassay of a protein using the monoclonal antibody of the present invention include an enzyme immunoassay, a radioimmunoassay, a fluorescence immunoassay and a luminescence immunoassay. In the enzyme immunoassay, the monoclonal antibody can be bound to an insoluble carrier to prepare a monoclonal antibody-bound insoluble carrier, and a sandwich enzyme immunoassay using the monoclonal antibody-bound insoluble carrier can also be performed.

(4) Protein Complex Containing the Protein of the Present Invention and mLin-10 The protein of the present invention binds to mLin-10 to form a complex and migrates on microtubules, thereby causing NMDA receptor 2B ( NR2B). The binding between the protein of the present invention (KIF17) and mLin-10 is considered to be the binding between the KIF17 tail domain and the first PDZ domain of mLin-10. Also, KI
It has been confirmed that the affinity between F17 and mLin-10 is about Kd = 3.0 × 10 ⁻⁶ M. The present invention also relates to a protein complex of the protein of the present invention (KIF17) and mLin-10.

(5) A method for screening for a substance that specifically interacts with the protein of the present invention or a transformant using the method of the present invention for identifying a substance that specifically binds to the protein of the present invention. A substance that specifically binds to the protein can be screened. As an example of such a method, C. Vries et al.
An expression cloning method that identified the EGF receptor is mentioned (Science, 255 , 989, 1992). The receptor can be used as a therapeutic agent for a disease associated with the protein of the present invention, or for research on a signal transduction system or biological function involving the protein of the present invention.

Further, a substance which controls the transcription of the gene encoding the protein of the present invention by coexisting the transformant of the present invention with various test substances and analyzing the expression level of the gene in the transformant, It is possible to screen for substances involved in the transcription control function of the protein of the present invention, or for genes that are subject to transcription control by the protein of the present invention.

(6) Pharmaceutical containing the protein or DNA of the present invention as an active ingredient The protein of the present invention exists on microtubules and has an activity of binding to mLin-10 to transport NMDA receptor 2B (NR2B). Have. Therefore, the protein of the present invention or DNA encoding the same is expected to be useful for treatment or prevention of diseases accompanied by abnormalities in nerve cell function. The types of diseases associated with abnormalities of nerve cell function are not particularly limited. For example, cerebral nerve diseases such as senile dementia (including Alzheimer's disease), Parkinson's disease, and Huntington's chorea; Down syndrome; neuropathy after cerebral infarction Neuropathy associated with head trauma; neurological disorders such as hydrocephalus; hereditary motor and sensory neuropathy; polyneuropathy of diabetes mellitus; age-related atrophy of peripheral sensation and motor nerves; Atrophy; age-related atrophy of neurons of the central nervous system; alcoholic polyneuropathy and the like.

The medicament containing the protein or DNA of the present invention as an active ingredient may be administered alone, but usually the active ingredient is combined with one or more pharmacologically acceptable carriers. And provided as a pharmaceutical formulation prepared by methods well known to those skilled in the art. A sterile solution dissolved in an aqueous carrier such as water or an aqueous solution (including salt, glycine, glucose, human albumin, etc.) is used. Further, pharmacologically acceptable additives (for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, sodium citrate, etc.) of the buffering agent and the tonicity agent can also be added.

The administration route is preferably the one that is most effective in the treatment, and may be oral administration or parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous. it can. Administration forms include sprays, capsules, tablets, granules, syrups, emulsions,
Suppositories, injections, ointments and the like can be mentioned. Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 1 μg / kg to 100 mg / kg per day for an adult. The present invention will be specifically described with reference to the following examples, but the present invention is not limited by the examples.

[0046]

EXAMPLES Example 1: Identification of KIF17 Factors belonging to the kinesin superfamily (KIF) include:
Supports diverse and well-regulated transport systems within cells (N. Hirokawa, Science 279, 519 (1998); and DLFole
tti et al., Neuron 23, 641 (1999)). To investigate the motors involved in the distribution of various molecules inside neurons, highly polarized cells with long axons and long dendrites, we first identified a novel neuron-specific motor molecule KIF17 was cloned as follows.

By screening a 4-week-old mouse brain cDNA library using the motor domain as a probe, 11 independent clones were obtained. When analyzed by Northern blot using a motor domain probe, a 5 kb KIF17 mRNA band was detected in the brain, a 3 kb band was detected in the testis, and the latter was KI.
It is thought to be a short mutant resulting from alternative splicing of F17.

Osm-3 (MAShakir et al., Neuroreport 4,891)
(1993); KIF17, a putative dendritic motor of odorant receptors in C. elegans, constitutes a novel family, similar to M. Tabish et al., J. Mol. Biol. 247, 377 (1995)). (FIG. 1B). KIF17 is similar to Osm-3 in the head and tail domains (FIG. 1C) and has an α-helical coiled-
It has two putative axial domains that form a coil structure. Osm-3, on the other hand, has only one axis domain. Phylogenetic trees, sequence comparisons, and prediction of coiled-coil structure were analyzed as previously reported (Y. Okada et al., Cell 81: 769. (1995); and N. Sa).
ito et al., Neuron 18,425 (1997)).

An antibody (αKIF17) against amino acids 505-507 of KIF17 (preparation and use is as follows)
In the brain, the native KIF17 protein is 170 kD in SDS-PAGE.
(FIG. 1E). Antibody (αKIF1
7) is amino acid 505 of KIF17 thioredoxin fusion protein
-717 (oblique bold in FIG. 1A). In all experiments in this example, purified full-length recombinant KIF17 protein (full-length KIF17-expressing baculovirus with His at the amino terminus was purified using the Fast-Bac system (Gibco Life Technol.
ogies). High-Five (Invitrogen) cells as previously reported (Y. Okada et al., Cell 81: 769. (1995); and
N. Saito et al., Neuron 18, 425 (1997)), Grace Insect
ΑKIF17 that was affinity-purified using Medium (Gibco Life Science), which had been infected with the virus for 60 hours, collected and purified.

In the Western blot of FIG. 1E, the brain sample was prepared using RIPA buffer (50 mM Tris, 1% Triton X-1).
00, 0.1% SDS, 150 mM NaCl, pH8.
Using 1 μg / lane of all the homogenate in 0), αKI
Detected at F17. αKIF17 is biotinylated kit (Pierce)
And avidin ALP (Pi
erce). 10 μg / ml purified recombinant KIF17
Was used to block the peptide. Note that αmLin-10 used in the following examples was obtained from Dr. Sudhof, and αmLin-7 was H
From Dr. ata (M.Irie et al., Oncogene 18, 2811 (199
9)), αNR1 (αGluRζ1No43), αNR2BC terminal peptide (αGluRεNo34) and αNR2BN terminal peptide (αGluRεNo59) (M. Watanabe et al., Eu
ro.J.Neurosci 10, 478 (1998); T.Kuttsuwada et al., Neuron
16, 333 (1996); and H. Mori et al., Neuron 21, 571 (199
8)) was obtained from Dr. Mishina.

In addition, monoclonal antibodies against mAb such as mLin-10, PDS-95, NR2B, synaptotagmin and mLin-2 (mA
bs) was purchased from the Transduction Laboratory and αsubst
The anceP receptor was purchased from Novus and the monoclonal (NR1 mAb) for α-glycine receptor and NA1 was purchased from Chemico
nGA and αGABA _A Rβ2 were purchased from Santa Cruz. Detection of proteins in the immunoprecipitant was performed using all available antibodies: mLin-10 antibody, NR1 antibody (αGluRζ1No43 and NR1 mAb), and NR2B antibody (αGluRεNo3).
4, αGluRεNo59, NR2B mAb).
The data shown in FIGS. 4 and 5 are data obtained by the monoclonal antibody.

KIF can be a monomer, homodimer, heterotrimer, homotetramer or heterotetramer (N.Hiro
kawa, Science 279, 519 (1998); and DLFoletti et al., Ne.
uron23, 641 (1999)), and biochemical analysis of KIF17 revealed that native KIF17 is a homodimeric motor protein without a binding polypeptide. Since the sedimentation coefficient is 3.0 S and the Stoke diameter is 170 Å, the molecular weight of the natural KIF17 holoenzyme is 215
kD, which is twice the molecular weight calculated from the protein sequence. The movement of KIF17 in native PAGE was similar to that of the recombinant full-length KIF17 protein.
These results are consistent with the fact that KIF17 exists in a homodimeric form. These experiments were performed in the following procedure.

The supernatant from the mouse brain homogenate was treated with a protease inhibitor (1 mM phenylmethylsulfonyl fluoride, 210 μM leupeptin, 145 μM pepstasin A, 260 μM N-α-p-tosyl-L-arginine methyl ester hydrochloride, And HBS containing 10 mM benzamidine (10 mM Hepes, pH 7.0).
4, 150 mM NaCl) in a Beckman TL100.3 rotor for 1 hour at 75000 rpm, followed by a speed gradient in a Beckman SW28 rotor. Gel filtration assay was performed using Superose 6 (Amersham Pharmacia Biotec).
h). Native PAGE was performed as previously reported (Y. Okada et al., Cell 81: 769. (1995); and N. Sait
o et al., Neuron 18, 425 (1997)).

To determine the motility and rate of KIF17 motor activity, motility was assayed using recombinant KIF17. Axial and microtubule motility assays for KIF17 consist of motility buffer (10 mM imidazole, 5 mM MgC
l ₂ , 1 mM EGTA, 5 mM DTT, 5 mM M
g-ATP, 50 mM NaCl, pH 7.4) was performed as described previously (Y. Okada et al., Cell 81: 7).
69. (1995); and N. Saito et al., Neuron 18, 425 (1997)). K
Glass surfaces with the IF17 tail construct (1-938) had similar motility in this assay. The KIF17 tail (1-938) and head (939-1038) mixture also has the same activity. These deletion mutants were assayed as positive controls in vesicle motility and competition assays.

As a result of the assay, KIF17 was found to be a microtubule-associated motor molecule that moves at a high speed toward the positive terminal. Recombinant KIF17 was able to slide the axon towards the minus end of the microtubule, indicating that KIF17 is acting as a motor towards the plus end of the microtubule (FIG. 1F). KIF1 by microtubule motility assay
The average speed of 7 was found to be 0.8-1.2 μm / sec. That is, it can be said that KIF17 acts without a coenzyme and mediates fast intramolecular transport.

Example 2: KIF17 is a dendritic-specific motor
-Protein: Tissue distribution analysis was performed as follows.
All homogenates of 4-week-old male ICR mice in RIPA were used and 20 μg of protein was used per lane. For neuron distribution analysis, 5 per lane
μg of protein was used. The results demonstrated that KIF17 was brain-specific, abundant in gray matter, especially hippocampus, and absent in white matter such as in the optic nerve (FIG. 2A).
Therefore, KIF17 is more likely to act in the cytoplasmic dendritic region of neurons than in axons.

Next, immunohistochemical analysis was performed as follows. The brains of 4-week-old male mice were fixed in 4% paraformaldehyde, 0.1% glutaraldehyde and 4% sucrose in PBS, and sections were prepared as previously reported (Y. Okad).
a et al., Cell 81: 769. (1995); and N. Saito et al., Neuron 18,
425 (1997)). Cultured hippocampal neurons were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The sample was blocked with 10% BSA and αKIF17 (5 μg
/ Ml) and a monoclonal antibody against phosphorylated neurofilament H (sigma, 1: 1000 dilution) or αM
Incubated with AP2 (sigma, 1: 1000 dilution). As a control, pre-immune IgG was used as the primary antibody. After overnight incubation at 4 ° C,
Samples were washed with PBS and incubated with Alexa488-conjugated goat antibody to rabbit IgG, and 10% BSA, mouse I
Alexa568 conjugated goat antibody to gG + H (Molecular Pr
obes 1: 100 dilution). TOTO-3 (Molecula
r Probes) were used as nucleotide markers for triple staining. For double staining with polyclonal antibodies in FIGS. 6B and C, detection was performed using Alexa568 streptavidin (Molecular Probes) using biotinylated αKIF17 according to the manufacturer's instructions. The sample was observed with a confocal laser scanning microscope LSM510 (Zeiss).

As a result, in the brain section immunostained with αKIF17, KIF17 was abundantly present in the dendrites of pyramidal neurons, but was found in the cerebral cortex (FIG. 2B), hippocampus (FIG. 2C), and olfactory bulb (FIG. 2D). This indicates that it is not present in axons or nuclei. Double staining of cultured hippocampal neurons with αKIF17 and an axon marker (anti-phosphorylated NF-H antibody)
F17 was found to be a dendritic motor (Fig. 2
E). Osm-3 is also known as a molecular motor localized to dendrites of specific neurons in C. elegans (MAShakir et al., Neuroreport 4,891 (1993); M. Tabish
Biol. 247, 377 (1995)).

Example 3: KIF17 transports transport material on membrane
To: To identify the transporters transported by KIF17, the structures that bind to KIF17 were examined by immunoisolation. Floating assays and EM studies were performed according to published methods (Y. Okada et al., Cell 81: 769. (1995); and N. Saito et al., Neur.
on 18, 425 (1997)). IM-Ac (Y. Okada et al., Cell 8
1: 769. (1995); and N. Saito et al., Neuron 18, 425 (1997))
The post-nuclear supernatant of the gray matter in the buffer was washed with Beckman NVTi65 at 65000 rpm for 4 hours at 40% to 0% Nycodentz.
, And 32 fractions were collected. Highest concentration
Fraction (26th) (~ 0.3 ml) containing KIF17 was
Incubated overnight at 4 ° C. with g of αKIF17 or 1 μg of control pre-immune IgG, then 12 hours at 4 ° C. with 10 μl of secondary antibody-coated magnetic beads (Dynal). The sample was then washed five times and further analyzed.

Observation of the intracellular organelles purified together with KIF17 by an electron microscope revealed clear membrane vesicles having an average radius of 50 nm (FIG. 3A). These vesicles were distinct from those transported by KIFC2, a CNS-specific dendritic motor protein (Y. Okada et al., Cel.
l 81: 769. (1995); and N. Saito et al., Neuron 18, 425 (199
7)). These membrane intracellular organelles were distinguishable from mitochondria, synaptic vesicles, dense core vesicles and multivesicular bodies. Next, the association between KIF17 and vesicles was examined biochemically. 1 μm of the fraction (100 μl) containing the floating vesicles
diluted with each buffer and gently agitated at 4 ° C. for 1 hour.
Incubate for 0 min and 10,000 g for 30 min at 4 ° C.
Pelletized by centrifugation for minutes. Control buffer; IMAc (pH 7.4), high salt buffer; 0.5
IMAc with M KCl, high pH buffer 0.1 M N
a ₂ CO ₃ (pH11.5).

As a result, the KIF17 molecule was released from the vesicle at a high salt concentration or a high pH (FIG. 3B). The results indicate that KIF17 is not embedded in the membrane,
It is suggested that there is a mechanism for the recognition of specific vesicles.

Example 4: mLin-10 is present on KIF17 present on the membrane.
Is a receptor for: yeast two-h using the C-terminal region of KIF17
Adapter molecules were identified by using ybrid screening. The yeast two-hybrid screening and assay was performed using the EGY48 yeast strain with Leu2 and β-galactosidase as reporter genes.
Performed as described in the standard protocol for ch.
KIF17 (939-1038) was subcloned into pLexA (LexA fusion vector), and pB42AD (G
〜1 × 10 ⁷ clones derived from a mouse brain cDNA library constructed in an AL1 activation domain vector (Clontech) were screened. 20 Leu ⁺ / Lac
Z ⁺ colonies were selected. Two colonies encode a fragment (457-840) of the mLin-10 gene, which is
It encodes a region that is 99% identical to the corresponding part of Lin-10 (Mint1). The cDNA fragment is amplified by PCR using specific primers, and pLexA or pB42AD
Was subcloned.

The KIF17 tail bound to the C-terminal PDZ domain of mLin-10, a partitioning protein. The binding partner of the PDZ domain of mLin-10, which is highly conserved from C. elegans to human, is unknown.

Further, the specificity of the binding between a number of KIFs and mLin-10 was examined using a mutation-containing prey. Binding is KI
It was very specific between the F17 tail domain and the first PDZ domain of mLin-10. Disruption of the first PDZ domain reduced binding to KIF17, whereas disruption of the second PDZ domain or deletion of the phospholipid interaction (PI) domain did not alter the interaction (FIG. 4A). KIF1
Substitution or deletion of one amino acid at the carboxyl terminus of 7 reduced the interaction (FIG. 4B). The PDZ domain tends to recognize proteins at its carboxyl terminus, which also applies to the binding of KIF17 to mLin-10. KI
F1A (400-terminal), KIF1B (830-terminal), KI
F5A (806-terminal), KIF5B (808-terminal), KI
As a result of testing the tail of F5C (930-terminal) for binding to mLin-10, only the tail of KIF17 bound to mLin-10.

Further, this coupling was examined by surface plasma resonance measurement (FIG. 4C). First, KIF17 (939-103)
8) The purified GST fusion C-terminus was replaced with BIACore CM5
Immobilized on a sensor chip, equilibrated with 50 mM Hepes-NaOH (pH 8.0) containing 100 mM NaCl, and purified with a flow rate of 10 μl / min.
650-840).
Binding activity (in the resonance unit) was measured as the difference between the basal value measured 20 seconds before sample injection and the measurement at the indicated time point. All experiments were performed at 25 ° C. using BIACORE3000. Data are from M. Irie et al., Science 277, 151.
1-5 (1997), BIA evaluation program 3.0 (B
IACORE).

As a result, it was confirmed that direct interaction between KIF17 and mLin-10 had a strong affinity (Kd = 3.0 × 10 ⁻⁶ M). This binding was strong enough to mediate partitioning in vivo.

This interaction was examined by pull-down assay and immunoprecipitation. The pull-down assay was performed using HBST (1
0 mM Hepes, pH 7.4, 150 mM NaC
l, 0.1% Triton X-100 and proteinase inhibitor), post-nuclear supernatant from 2 week old mouse brain lysate and 10 μg of immobilized GST-fused KIF17 tail region recombinant protein and KIFC2 1-433 This was performed using the protein. After 12 hours incubation, 5 beads
Washed twice and immunoblotted with an antibody against mLin-10.
The recombinant protein was E. coli strain BL21 (DE3) (strarage
ne) into a pGEX vector (Amersham Pharmacia Biotec)
h) and 10 μl of Glutachione Sepharo
Purified by se Fast Flow (Amersham Pharmacia Biotech).

As a result, mLin-10 was pulled down together with the recombinant KIF17, but not with KIFC2 or KIF17 in which the terminal amino acid was deleted (FIG. 4D).

Also, 4-week-old mouse brains were dissected as previously reported in HBST buffer (Y. Okada et al., Cell 81: 769. (19)
95); and N. Saito et al., Neuron 18, 425 (1997)). 0.5 ml of brain lysate was added to 20 μl of αmLin-10 serum and 10 μl of protein A Sepharose Fast Flow (Amer
(sham Pharmacia Biotech) at 4 ° C. overnight and washed 5 times with 0.5 ml HBST. 5μ
1 / lane of sample was added. The control used was normal rabbit IgG. As a result, mLin-10 and KIF17
Simultaneously immunoprecipitated, but other known KIFs (KIFC2, KIF1
A, KIF3A / B, KIF5A / B / C) could not be detected in the immunoprecipitates. The results confirmed the specificity of the interaction between mLin-10 and KIF17 (FIG. 4E). From these results, mLin
-10 is confirmed to be a specific binding partner of KIF17 in vivo.

Example 5: Operated by KIF17 / mLin-10 complex
NR2B distribution vesicles carried: C. elegan with mutation in Lin-10
In G.s, the glutamate receptor GluR1 is mislocalized (JSSimske et al., Cell 85,195 (1996);
S. Bredt, Cell 94, 773 (1998); JPBorg et al., J. Biol. Che
m. 273, 31633 (1998); and SMKaech et al., Cell 94, 761.
(1998)). mLin10 is present in a protein complex with mlin-2 and mLin-7, which binds to the glutamate receptor subunit NR2B (K. Jo. et al., J. Neurosci. 19, 4189 (199
9)). To determine whether mLin-2, mLin-7 and NR2B are transporter molecules of the KIF17-mLin-10 complex, the developmental expression patterns of transporter proteins were examined. First, whole brain homogenates were prepared from 11 dpc fetuses to 3 week old newborns. A sample of 20 μg / lane was loaded on SDS-PAGE, and each protein was detected by Western blotting.
As a result, the expression of the candidate protein was consistent with the expression of KIF17 (FIG. 5A), but ordinary kinesin (KIF5B) showed a different expression pattern.

KIF17, mLin-10, mLin-2 and mLin on vesicles
-7 floated as NR2B in the same low-density membrane protein fraction, but did not float in other membrane protein fractions containing vesicles or mitochondrial cox. Liberated KIF17, mLin-10,
mLin-2 and mLin-7 were collected in the heavier fraction. The ratio of free protein to membrane-bound protein depends on the expected binding order of protein to membrane (KIF17, mLin-10, mLin-2, mLin-
7, NR2B) (FIG. 5B) (FIG. 5B shows fractions from the flotation assay described above).

The immunoprecipitation using αKIF17 was performed as follows. Four-week-old male mouse brains were placed in HBS buffer as previously reported (Y. Okada et al., Cell 81: 769. (1995); and N. Saito et al., N.
euron 18, 425 (1997)). Brain lysate
5 ml were incubated overnight at 4 ° C. with 20 μl αKIF17 serum and 10 μl protein A Sepharose FastFlow and washed 5 times with 0.5 ml HBST. 5μ
1 / lane of sample was added. NR2B is less than 5% in 0.1% triton, less than 5% in RIPA, 3% in 1% Doc.
Solubilized by less than 0% (Jaroslav Blahos et al., J. Biol.
Chem. 271: 15669-15674 (1996)). The amount of NR2B immunoprecipitated by KIF17 was 5% of the lysate in 0.1% triton.
Below, 5% or less of lysate in RIPA, 1% Doc
(Jaroslav Blahos et al., J. Biol. Ch.
em.271: 15669-15674 (1996)). At least two types of NR2B
May be present, one is taken up and anchored in the postsynaptic membrane region, the other is present in cytoplasmic vesicles, some of which are transporting complexes with KIF17 and mLin 10/7/2 May have formed. In this example, 0.1% triton was selected because strong detergent treatment may affect the preservation of cytoplasmic NR.

As a result of the immunoprecipitation assay, mLin-10, mLin-
2, mLin-7, detected with antibodies to NR2B receptor,
It was not detected with an antibody against PSD-95 that binds to NR2B in postsynaptic membrane thickening (FIG. 5C). GABA _A receptor β2, glycine receptor and substance P receptor were not detected (Jaroslav Blahos et al., J. Biol. Chem. 2
71: 15669-15674 (1996)). KIF17 motor, mLin-10, mLin-2, mLin-7, and NR in immunoprecipitation using αmLin-10
2B interaction was confirmed (FIG. 4E). NR2B, mLin-1
0, mLin-2 and mLin-7 are detected by αKIF17 in isolated vesicles, but not αKIF1A (FIG. 5).
C).

Since transport is a transient state of NR2B, the ratio of immunoprecipitate to total is less with NR2B than with the other components of the complex. Some of the vesicles containing NR2B bind to microtubules in a nucleotide-dependent manner, and KIF17 co-localizes to these vesicles (FIG. 5D), binding of NR2B and vesicles to microtubules. Is the KIF17 tail peptide (GST-KIF
17 939-1038) (FIG. 5).
E). This peptide does not inhibit the MT binding activity of the KIF17 motor domain (described above), but binds to native mLin10 (described above). Thus, it can be concluded in this assay that the KIF17 tail peptide competes with native KIF17 to bind to mLin10 and reduce the number of NR2Bs containing transporters on microtubules. These data suggest that these proteins form a protein complex on the vesicle.
This assay was performed as follows.

Vesicle immunoprecipitation is performed as previously reported (Y. Okad).
a et al., Cell 81: 769. (1995); and N. Saito et al., Neuron 18,
425 (1997)). An additional band detected on its own on the immunoprecipitated product of KIF1A is a degradation product that appears after incubation. 2mg / ml BSA coated with microtubules
10 μl cover glass chamber blocked with 5
The mixture was incubated with the floating fraction supplemented with mM AMP-PNP or ATP at 27 ° C for 10 minutes, washed with each buffer, and immunodetected as described above. Vesicles on microtubules are D
Found using IC-LSM. For the blocking assay, the KIF17 tail peptide (GST-KIF1793) was used.
9-1038) was incubated with the floating vesicles for 6 hours before the microtubule binding assay was performed. The total protein concentration was equalized by the addition of GST alone.

Example 6: KIF17 in the same dendrite
The co-localized brain section of -mLin10-NR2B and the cultured hippocampal neurons were immunostained by the following procedure. Mouse hippocampal neurons were fixed in 4% PFA,
Infiltrated with 0.1% Triton X-100 for 3 minutes. 4 brain slices
Fixed in% PFA. To stain the sections with NR2B,
Pepsin treatment was performed as previously reported (M. Watanabe et al., Euro.
J. Neurosci. 10, 478 (1998); T. Kutsuwada et al., Neuron 16,
333 (1996); and H, Mori et al., Neuron 21,571 (1998)). αNR
The same result was obtained with the combination of 1 (αGluRζ1No43) and NR1 mAb, the C-terminal peptide of αNR2B (αGluRεNo34) and the N-terminal peptide of αNR2B (αGluRεNo59). As described above, double staining using antibodies against αKIF17 and NR1, NR2B, and mLin-10 revealed the co-localization of these proteins in the same dendrites (FIG. 6).

Example 7: Visualization of NR2B transport by KIF17 In this experiment, first the membrane organelles carrying KIF17 were purified from the KIF17-rich fraction of the suspension assay. 5 μl of αK
IF17 immunobeads were incubated with the vesicles for one hour under three different conditions. (1) 30 μl of 1 mg / m
(2) 1 mg / ml recombinant tail-deficient KIF17 (1-938); (3) 0.2 mg / m
1 tail construct (GST-KIF17 939-1038)
0.8 mg / ml tail-deficient construct (1-9
38). Through this process, KIF17 vesicles are released from the beads. In condition (3), the addition of the tail construct ruled out the possible difference in vesicle release from the beads. The eluted supernatant was then diluted in 100 μl motility buffer (described above) and poured into the observation chamber. Under all three conditions, Brownian motion of the released vesicles was observed. In condition (1), almost all vesicles in contact with the shaft thread (more than 10 movements in 3 minutes of observation)
Moves. Under conditions (2) and (3), no movement of vesicles on the axial thread was observed. The identification of vesicles correlated to motility has been described in the literature (Jack Taunton et al., J. Cell. Biol. 148: 519-53).
0 (2000)). Less than 40% of the vesicles bound to microtubules by KIF17 were NR2B positive. NR2B was not detected in vesicles isolated using anti-KIF3B immunobeads as a control.

As a result of the above experiment, natural vesicles purified from the brain using αKIF17 beads were found to have a KIF17 full length of 1-1038.
When incubated with, they migrated in the direction toward the plus end on microtubules (FIGS. 7A, B). mLin-10
KIF17 deletion recombinant without interaction domain (1-
938) do not move these vesicles on microtubules, only Brownian motion of vesicles in buffer is observed. This deletion mutant has motor activity in a microtubule motility assay,
Since the vesicles are indeed released, this result indicates that the mLin-10 binding site is required for KIF17 to transport these vesicles. Immobilization directly and immunodetection by fluorescence in LM (FIG. 7C) and gold label in EM (FIG. 7D) revealed that NR2B was present on these active vesicles. From these results, NR2B
It is concluded that the carrier containing is transported by KIF17 and that the mLin10 binding tail is required for this function of KIF17.

The above data indicate that KIF17, a neuron-specific molecular motor with motility oriented toward the plus end of microtubules, directly interacts with the mLin-10PDZ domain to transport NR2B to dendrites. It suggests that.

[0080]

According to the present invention, a novel microtubule-associated motor protein has been identified. The identification of the protein of the present invention has elucidated a part of the mechanism of transport of neurotransmitter receptors, and has provided useful information for the development of a medicament for treating, preventing and / or diagnosing a nerve-related disease. Can be

[0081]

[Sequence List] <110> Nobutaka HIROKAWA <120> A Kinesin Superfamily Motor Protein KIF17 <130> <160> 3

<210> 1 <211> 1038 <212> PRT <213> human <400> 1 Met Ala Ser Glu Ser Val Lys Val Val Val Arg Cys Arg Pro Met Asn 1 5 10 15 Lys Arg Glu Arg Glu Leu Ser Cys Gln Ser Val Val Thr Val Asp Ser 20 25 30 Ala Arg Gly Gln Cys Phe Ile Gln Asn Pro Gly Ala Ala Asp Glu Pro 35 40 45 Pro Lys Gln Phe Thr Phe Asp Gly Ala Tyr Tyr Ile Glu His Phe Thr 50 55 60 Glu Gln Ile Tyr Asn Glu Ile Ala Tyr Pro Leu Val Glu Gly Val Thr 65 70 75 80 Glu Gly Tyr Asn Gly Thr Ile Phe Ala Tyr Gly Gln Thr Gly Ser Gly 85 90 95 Lys Ser Phe Thr Met Gln Gly Leu Pro Asp Pro Pro Cys Gln Arg Gly 100 105 110 Ile Ile Pro Arg Ala Phe Glu His Val Phe Glu Ser Val Gln Cys Ala 115 120 125 Glu Asn Thr Lys Phe Leu Val Arg Ala Ser Tyr Leu Glu Ile Tyr Asn 130 135 140 Glu Asp Val His Asp Leu Leu Gly Ala Asp Thr Lys Gln Arg Leu Glu 145 150 155 160 Leu Lys Glu His Pro Glu Lys Gly Val Tyr Val Lys Gly Leu Ser Met 165 170 175 His Thr Val His Asn Val Ala Gln Cys Glu Arg Val Met Glu Thr Gly 180 185 190 Trp Lys Asn Arg Ala Val Gly Ty r Thr Leu Met Asn Lys Asp Ser Ser 195 200 205 Arg Ser His Ser Ile Phe Thr Ile Asn Ile Glu Ile Tyr Ala Val Asp 210 215 220 Glu Arg Gly Lys Asp His Leu Arg Ala Gly Lys Leu Asn Leu Val Asp 225 230 235 240 Leu Ala Gly Ser Glu Arg Gln Ser Lys Thr Gly Ala Thr Gly Glu Arg 245 250 255 Leu Lys Glu Ala Thr Lys Ile Asn Leu Ser Leu Ser Ala Leu Gly Asn 260 265 270 Val Ile Ser Ala Leu Val Asp Gly Arg Cys Lys His Ile Pro Tyr Arg 275 280 285 Asp Ser Lys Leu Thr Arg Leu Leu Gln Asp Ser Leu Gly Gly Asn Thr 290 295 300 Lys Thr Leu Met Val Ala Cys Leu Ser Pro Ala Asp Asn Asn Tyr Asp 305 310 315 320 Glu Thr Leu Ser Thr Leu Arg Tyr Ala Asn Arg Ala Lys Asn Ile Lys 325 330 335 Asn Lys Pro Arg Ile Asn Glu Asp Pro Lys Asp Ala Leu Leu Arg Glu 340 345 350 Tyr Gln Glu Glu Ile Lys Arg Leu Lys Ala Ile Leu Ala Gln Gln Met 355 360 365 Gly Pro Gly Asn Leu Ser Ala Leu Leu Ser Thr Gln Thr Pro Pro Gly 370 375 380 Pro Val Gln Ser Glu Glu Lys Leu Leu Ser Pro Thr Thr Val Gln Gln 385 390 395 400 Asp Thr Glu Ala Glu Lys Gln Le u Ile Arg Glu Glu Tyr Glu Glu Arg 405 410 415 Leu Ala Arg Leu Lys Ala Asp Tyr Glu Ala Glu Gln Glu Ser Arg Val 420 425 430 Arg Leu Gln Glu Asp Ile Thr Ala Met Arg Asn Ser Tyr Asp Val Lys 435 440 445 Leu Ser Thr Leu Gln Glu Asn Leu Arg Lys Glu Lys Glu Thr Glu Ala 450 455 460 Ile Leu Lys Ala Glu Val Leu Cys Lys Thr Glu Val Met Ser Arg Ala 465 470 475 480 Glu Leu Ala Ser Gly Pro Glu Tyr Ser Pro Pro Leu Gln Tyr Glu Thr 485 490 495 Ala Val Lys Pro Thr Ile Leu Ser Met Pro Asp Met Pro Pro Ser Gly 500 505 510 Lys Val Thr Lys Ser Gln Ala Pro Leu Ala Phe Glu Glu Pro His Gly 515 520 525 Glu Thr Ser Arg Ser Glu Phe Ser Phe Glu Ser Asn Glu Cys Ser Thr 530 535 540 Leu Glu Asp Ser Ala Thr Ser Glu Ala Phe Pro Gly Pro Glu Glu Phe 545 550 555 560 Ser Asn Met Glu Phe Ser Met Ala Ala Ala Leu Thr Glu Ser Arg Tyr 565 570 575 Leu Pro Glu Glu Tyr Leu Gly Gly Gln Glu Ala Ala Ala Ser Pro Leu 580 585 590 Glu Ala Glu Arg Tyr Val Gln Glu Asn Glu Pro Ser Leu Glu Pro Leu 595 600 605 Arg Ile Leu Ala Ser Leu Gln Asp Pr o Phe Ala Glu Val Glu Ala Lys 610 615 620 Leu Ala Arg Leu Ser Ser Thr Val Ala Met Ser Asp Ser Ser Gln Thr 625 630 635 640 Val Val Pro Gln Ile Pro Lys Gln Pro Ser Ser Ala Asp Leu Leu Glu 645 650 655 Pro Ser Asp Thr Lys Ser Glu Ala Asp Val Ala Val Ala Asp Asn Val 660 665 670 Val Leu Gly Thr Glu Pro Asp Val Asn Leu Arg Val Ala Glu Glu Val 675 680 685 Val Ser Glu Ala Glu Thr Gly Val Trp Met Glu Ser Glu Ala Gln Val 690 695 700 Ala His Val Ala Gln Val Ser Glu Glu Ala Gln Pro Gln Pro Leu Leu 705 710 715 720 Ala Met Val Ser Val Arg Arg Glu Ser Val Gly Val Glu Val Ala Val 725 730 735 Leu Thr Glu Glu Glu Leu Gln Pro Val Asp Gln Gln Gln Val Leu Ala 740 745 750 Arg Leu Gln Leu Leu Glu Gln Gln Val Val Gly Gly Glu Gln Ala Lys 755 760 765 Asn Lys Asp Leu Arg Glu Lys His Lys Arg Arg Lys Arg Tyr Ala Asp 770 775 780 Glu Arg Lys Lys Gln Leu Val Ala Ala Leu Gln Asn Ser Asp Glu Asp 785 790 795 800 Gly Gly Asp Trp Val Leu Leu Asn Val Tyr Asp Ser Ile Gln Glu Glu 805 810 815 Val Arg Ala Lys Ser Lys Leu Leu Leu Gl u Lys Met Gln Arg Lys Leu Arg 820 825 830 Ala Ala Glu Val Glu Ile Lys Asp Leu Gln Ser Glu Phe Gln Leu Glu 835 840 845 Lys Ile Asp Tyr Leu Ala Thr Ile Arg Arg Gln Glu Arg Asp Ser Met 850 855 860 Leu Phe Gln Gln Leu Leu Glu Gln Val Gln Pro Leu Ile Arg Arg Asp 865 870 875 880 Cys Asn Tyr Ser Asn Leu Glu Lys Ile Arg Arg Glu Ser Ser Trp Asp 885 890 895 Glu Asp Asn Gly Phe Trp Lys Ile Pro Asp Pro Ile Ile Leu Lys Thr 900 905 910 Ser Leu Pro Val Val Pro Thr Gly Thr Gln Asn Lys Pro Ala Arg Lys 915 920 925 Thr Ser Ala Val Asp Ser Gly Glu Pro His Met Gln Glu Glu Asp Arg 930 935 940 Tyr Lys Leu Met Leu Ser Arg Ser Asp Ser Glu Asn Ile Ala Ser Asn 945 950 955 960 Tyr Phe Arg Ser Lys Arg Ala Ser Gln Ile Leu Ser Thr Asp Pro Met 965 970 975 Lys Ser Leu Thr Tyr His Asn Ser Pro Pro Gly Leu Asn Ser Ser Leu 980 985 990 Ser Asn Asn Ser Ala Leu Pro Pro Thr Gln Thr Pro Glu Met Pro Gln 995 1000 1005 Pro Arg Pro Phe Arg Leu Glu Ser Leu Asp Ile Pro Phe Ser Lys Ala 1010 1015 1020 Lys Arg Lys Lys Ser Lys Asn Ser P he Gly Gly Glu Pro Leu 1025 1030 1035

[0083] <210> 2 <211> 3432 <212> DNA <213> human <400> 2 gtcgtgcggc ggcgcctgca gcgcggatcc cccgttcccc gcccgggccc ggcttcaggg 60 acgggcgagc ccgggcgcgg gaggcggcgg cccgggttcc agcccgcaca tttgtccgcc 120 gagcccttgc cacacccagt gcgccccgcg gggagtcccg gccccttggt gcggccccgc 180 ccccgccgcc aggccccgcc ccgcgcgggt ctggctccac ccctggaccg ggccacgccc 240 ccgggcagcc atggcttcgg agtcagtgaa agtggtcgtg cgctgccgac ccatgaacaa 300 gcgcgagcgc gagctgagct gccagtcggt ggtgacggtg gacagtgcgc gcggccaatg 360 cttcattcag aacccgggag cggcggacga acccccaaag cagttcacct tcgatggcgc 420 ctactacata gaacacttca ccgagcagat ctacaacgag atcgcctacc cgctggtgga 480 gggtgtcacc gaaggctaca atgggaccat ttttgcctat ggccagacag gcagtgggaa 540 gtctttcacc atgcagggac tgccagatcc cccctgccag aggggcatca ttcccagagc 600 cttcgagcac gtgtttgaga gcgttcagtg tgcagagaac accaagttct tagtgcgggc 660 ttcctatctg gagatctaca acgaagatgt ccatgacctg ctgggcgctg acaccaagca 720 gaggcttgag ctgaaggagc atcccgagaa gggagtgtat gtcaaggggc tgtccatgca 780 cacggtacac aacgtggccc a gtgcgagcg cgtcatggag accggctgga agaaccgtgc 840 ggttggctac accctgatga ataaggactc ctcccgttcc cactccatct ttaccatcaa 900 catcgagatc tatgccgtgg atgaacgggg taaggaccat cttcgtgcag gcaagctcaa 960 cctggtagac ctggccggca gtgagcggca atccaagact ggggccacag gggaaaggct 1020 gaaggaggcc accaaaatta acctgtcact gtcggcgctg ggcaacgtca tctcggcact 1080 ggtggatggg cgctgcaagc acattcctta ccgggactcg aagctgaccc gactgcttca 1140 ggactcactg ggtggcaaca ccaagactct gatggtggct tgcctgtctc ccgcagacaa 1200 caattacgat gagaccctca gtacgctgcg ctatgctaac agggccaaga acatcaagaa 1260 caagccacgc attaatgaag accccaagga tgccctgctt cgagagtacc aggaagagat 1320 caagaggcta aaggctatcc tcgcccagca gatgggccct ggcaacctgt cagccctgct 1380 gtccactcag acacccccag gcccagtcca gtctgaggag aagctgctgt ccccaactac 1440 tgtccagcag gacacggagg ctgagaagca gttgatccga gaggagtatg aggagcgcct 1500 ggcccgcctg aaggctgact acgaggcaga gcaagagtct cgggtccggc tgcaggagga 1560 cattactgcc atgcgtaact cgtatgacgt gaagctgtcc acactgcagg agaatctgcg 1620 caaggagaag gagacagagg ccattctcaa ggcggaagtc ctgtgtaaga cggaggtcat 1680 gtccagggcc gagttagcca gcgggcccga gtactcacct cctttgcagt atgagactgc 1740 ggtgaagccc accatcctct ccatgcccga catgccaccc agtggcaagg tcaccaagag 1800 ccaagcgccc ctcgctttcg aggaaccaca cggggagacc tccaggtccg agttttcctt 1860 tgagtccaat gagtgttcca ctctcgaaga ctctgccaca tcagaggcct tccctgggcc 1920 agaggagttt tccaacatgg agttctccat ggctgcggca ctgaccgaga gcaggtacct 1980 tcctgaggag tatctcggcg gccaggaggc tgctgcatct ccgctggagg cagagcgcta 2040 tgtccaggag aacgagccat cgctggagcc cctgcggata ctggcaagcc tgcaggatcc 2100 ttttgctgaa gtggaggcca agctggccag actgtcctct actgtggcca tgtcggactc 2160 atcccagacc gtcgtccccc agatccctaa gcagccatcc tcggctgacc tgctggagcc 2220 cagcgacacc aagtcagaag ctgatgtggc tgtggctgac aacgtcgtcc tcgggactga 2280 gcctgatgta aacctgagag tggctgagga ggtggtttct gaggcagaga ctggcgtgtg 2340 gatggagtct gaagcccagg tggcccatgt ggcccaggtg tctgaggagg cccagcctca 2400 gcccttgctg gccatggtga gcgtgaggag ggaaagtgtg ggtgtagaag tggctgtgtt 2460 gacggaggag gagctgcagc ctgtggatca acagc aggtg ctggcccgct tgcagttgct 2520 ggagcagcag gtggtggggg gcgagcaggc taaaaacaag gacctgaggg agaagcacaa 2580 gcggcggaag cggtacgcag atgagcgcaa gaagcagctg gtggccgcac tgcagaactc 2640 ggatgaggat ggcggggact gggtgctgct caacgtctat gactctatcc aggaggaagt 2700 gagggccaag agcaagctgc tggagaagat gcagaggaag ctccgggcag cagaggtaga 2760 aatcaaggac ctgcagtcag agttccagct ggagaagatt gattacctgg caaccatccg 2820 ccggcaggag cgggactcca tgctcttcca gcaactcctg gagcaggtgc agcctctcat 2880 ccgcagagac tgtaactata gcaacctgga gaagatcagg cgagagtcga gctgggatga 2940 ggataatggc ttctggaaga tcccagatcc catcatactg aaaaccagcc tcccagtagt 3000 tccaactggg acacagaaca agccagctcg aaaaacctct gcagtagaca gtggggagcc 3060 acacatgcag gaggaagaca ggtataagct gatgctcagc cgaagcgaca gtgagaatat 3120 cgctagcaac tacttccgct ccaagcgggc cagccagatc ctcagcacgg accccatgaa 3180 gagcctcaca tatcacaact caccaccggg gctaaattcc tctctgagca acaactctgc 3240 cttgccgccc acccagaccc cggaaatgcc ccagccccgg cccttccgcc tagagtccct 3300 tgacatcccc ttctccaaag ccaagcgtaa aaaaagcaaa aacagcttcg gtggtgagcc 3360 tctgtgatcc agctgcctgc tgctatctgc tttcaagaaa cttccgagtc ctctctgcaa 3420 agccccaacc ag 3432

[0084] <210> 3 <211> 3425 <212> DNA <213> human <400> 3 atggcttcgg agtcagtgaa agtggtcgtg cgctgccgac ccatgaacaa gcgcgagcgc 60 gagctgagct gccagtcggt ggtgacggtg gacagtgcgc gcggccaatg cttcattcag 120 aacccgggag cggcggacga acccccaaag cagttcacct tcgatggcgc ctactacata 180 gaacacttca ccgagcagat ctacaacgag atcgcctacc cgctggtgga gggtgtcacc 240 gaaggctaca atgggaccat ttttgcctat ggccagacag gcagtgggaa gtctttcacc 300 atgcagggac tgccagatcc cccctgccag aggggcatca ttcccagagc cttcgagcac 360 gtgtttgaga gcgttcagtg tgcagagaac accaagttct tagtgcgggc ttcctatctg 420 gagatctaca acgaagatgt ccatgacctg ctgggcgctg acaccaagca gaggcttgag 480 ctgaaggagc atcccgagaa gggagtgtat gtcaaggggc tgtccatgca cacggtacac 540 aacgtggccc agtgcgagcg cgtcatggag accggctgga agaaccgtgc ggttggctac 600 accctgatga ataaggactc ctcccgttcc cactccatct ttaccatcaa catcgagatc 660 tatgccgtgg atgaacgggg taaggaccat cttcgtgcag gcaagctcaa cctggtagac 720 ctggccggca gtgagcggca atccaagact ggggccacag gggaaaggct gaaggaggcc 780 accaaaatta acctgtcact g tcggcgctg ggcaacgtca tctcggcact ggtggatggg 840 cgctgcaagc acattcctta ccgggactcg aagctgaccc gactgcttca ggactcactg 900 ggtggcaaca ccaagactct gatggtggct tgcctgtctc ccgcagacaa caattacgat 960 gagaccctca gtacgctgcg ctatgctaac agggccaaga acatcaagaa caagccacgc 1020 attaatgaag accccaagga tgccctgctt cgagagtacc aggaagagat caagaggcta 1080 aaggctatcc tcgcccagca gatgggccct ggcaacctgt cagccctgct gtccactcag 1140 acacccccag gcccagtcca gtctgaggag aagctgctgt ccccaactac tgtccagcag 1200 gacacggagg ctgagaagca gttgatccga gaggagtatg aggagcgcct ggcccgcctg 1260 aaggctgact acgaggcaga gcaagagtct cgggtccggc tgcaggagga cattactgcc 1320 atgcgtaact cgtatgacgt gaagctgtcc acactgcagg agaatctgcg caaggagaag 1380 gagacagagg ccattctcaa ggcggaagtc ctgtgtaaga cggaggtcat gtccagggcc 1440 gagttagcca gcgggcccga gtactcacct cctttgcagt atgagactgc ggtgaagccc 1500 accatcctct ccatgcccga catgccaccc agtggcaagg tcaccaagag ccaagcgccc 1560 ctcgctttcg aggaaccaca cggggagacc tccaggtccg agttttcctt tgagtccaat 1620 gagtgttcca ctctcgaaga ctctgccaca tcagaggcct tccctgggcc agaggagttt 1680 tccaacatgg agttctccat ggctgcggca ctgaccgaga gcaggtacct tcctgaggag 1740 tatctcggcg gccaggaggc tgctgcatct ccgctggagg cagagcgcta tgtccaggag 1800 aacgagccat cgctggagcc cctgcggata ctggcaagcc tgcaggatcc ttttgctgaa 1860 gtggaggcca agctggccag actgtcctct actgtggcca tgtcggactc atcccagacc 1920 gtcgtccccc agatccctaa gcagccatcc tcggctgacc tgctggagcc cagcgacacc 1980 aagtcagaag ctgatgtggc tgtggctgac aacgtcgtcc tcgggactga gcctgatgta 2040 aacctgagag tggctgagga ggtggtttct gaggcagaga ctggcgtgtg gatggagtct 2100 gaagcccagg tggcccatgt ggcccaggtg tctgaggagg cccagcctca gcccttgctg 2160 gccatggtga gcgtgaggag ggaaagtgtg ggtgtagaag tggctgtgtt gacggaggag 2220 gagctgcagc ctgtggatca acagcaggtg ctggcccgct tgcagttgct ggagcagcag 2280 gtggtggggg gcgagcaggc taaaaacaag gacctgaggg agaagcacaa gcggcggaag 2340 cggtacgcag atgagcgcaa gaagcagctg gtggccgcac tgcagaactc ggatgaggat 2400 ggcggggact gggtgctgct caacgtctat gactctatcc aggaggaagt gagggccaag 2460 agcaagctgc tggagaagat gcagaggaag ctccg ggcag cagaggtaga aatcaaggac 2520 ctgcagtcag agttccagct ggagaagatt gattacctgg caaccatccg ccggcaggag 2580 cgggactcca tgctcttcca gcaactcctg gagcaggtgc agcctctcat ccgcagagac 2640 tgtaactata gcaacctgga gaagatcagg cgagagtcga gctgggatga ggataatggc 2700 ttctggaaga tcccagatcc catcatactg aaaaccagcc tcccagtagt tccaactggg 2760 acacagaaca agccagctcg aaaaacctct gcagtagaca gtggggagcc acacatgcag 2820 gaggaagaca ggtataagct gatgctcagc cgaagcgaca gtgagaatat cgctagcaac 2880 tacttccgct ccaagcgggc cagccagatc ctcagcacgg accccatgaa gagcctcaca 2940 tatcacaact caccaccggg gctaaattcc tctctgagca acaactctgc cttgccgccc 3000 acccagaccc cggaaatgcc ccagccccgg cccttccgcc tagagtccct tgacatcccc 3060 ttctccaaag ccaagcgtaa aaaaagcaaa aacagcttcg gtggtgagcc tctgtgatcc 3120 agctgcctgc tgctatctgc tttcaagaaa cttccgagtc ctctctgcaa agccccaacc 3180 aggtcgacgc ggccgcgaat tcctgcagcc cgggggatcc actagttcta gagcggccgc 3240 caccgcggtg gagctccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg 3300 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 3360 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttccccaa 3420 cagtt 3425

[Brief description of the drawings]

FIG. 1 shows the identification of KIF17 as a homodimeric MT-dependent motor protein. (A) shows the total length of KIF17
038 shows the amino acid sequence (the amino acid sequence is described in SEQ ID NO: 1, the base sequence of the full-length DNA encoding it is described in SEQ ID NO: 2, and the base sequence of the ORF is described in SEQ ID NO: 3). The motor domain probes used for screening are shown in bold, the middle domain used for making the antibodies is shown in italics, and the tail domain used for two hybrid screening is shown in italics.
(B) shows the relationship between KIF17 and other known KIFs using a phylogenetic tree. (C) shows the results of protein matrix analysis between KIF17 and Osm-3, showing the similarity of their head and tail domains. (D) shows that the central region of KIF17 is α-
This shows that a helical coiled-coil structure is expected to form two stretches. (E) shows natural KIF1
7 shows the results of the immunoblot of FIG. Block: peptide block using recombinant KIF17 as a control Br: brain Liv: liver (F) shows the results of a kinetic assay of recombinant KIF17 using an axial thread. The arrow indicates the dispersed plus end of the bundle of axial microtubules.

FIG. 2 shows the localization of KIF17 in dendrites. (A) shows a KIF17 immunoblot in each tissue. Br (brain), Lu (lung), Ht (heart), Li (liver) Sp (spleen), Kid (kidney), Mu (muscle), O
B (olfactory bulb), CC (cerebral cortex), PU (putamen), Hip
(Hippocampus), Ce (cerebellum), Pons (bridge), Spi (spinal cord), CE (cauda equina), Sci (sciatic nerve), Opi (optic nerve), KIF5B was used as a control in the lower lane. (B) shows αKIF17, α-phosphorylated neurofilament H (pNFH) as an axon marker, and D
The pyramidal layer of the cerebral cortex triple-stained with TOTO-3 (NA) as an NA and RNA marker is shown. Scale bar is 1
5 μm. (C) Hippocampal CA3 triple stained with αKIF17, αMAP2 as a dendritic marker, and TOTO-3
Indicates the area. The bar indicating the scale is 15 μm. Peptide inhibition staining of αKIF17 as a negative control is shown in the upper small box. (D) shows αKIF17, αpNFH,
And Olfactory bulb mitral cell layer triple stained with TOTO-3. The bar indicating the scale is 15 μm. (E) shows cultured hippocampal neurons double stained with αKIF17 and αpNFH. The bar indicating the scale is 15 μm.

FIG. 3 shows the transporter of KIF17. (A) is αKIF
17 shows the EM of the vesicle immunoisolated at 17. The bar indicating the scale is 20 nm. The bar graph shows the number of vesicles per 100 beads. (B) shows the binding of KIF17 to vesicles assayed under different washing conditions.

FIG. 4 shows a direct interaction between the KIF17 tail and the mLin-10 PDZ domain. (A) and (B)
Is KIF17 and mLin-10 using yeast two-hybrid system
2 shows the identification of the interaction domain of. Amino acid residues of Lin-10 and KIF17 fused to the pLex DNA binding domain are indicated by squares and the corresponding amino acid positions are indicated. (+) Is KI
F17 and those bound to mLin-10 are shown, (-) indicates KIF17
And those that do not bind to mLin-10. ELGF is a known interacting consensus sequence of PDZ with the COOH terminus of the binding partner, ILGV (668 in mLin-10).
−671) → AAA represents the ELGF motif, and all were mutated to alanine by site-specific PCR to destroy the first PDZ motif. GLGF (7 of mLin-10)
60-763) → The AAAA clone is another mutant that disrupted the second PDZ motif. KIF17 delta 3 amino acids are KIF17 with three COOH terminal amino acids deleted.
The construction of KIF17EPL ^* → EPA ^* is wild type C
Shown is a construct in which the OOH terminal sequence EPL stop codon was mutated to an EPA stop codon. EPL ^* → EAL ^* , EPL ^*
→ EAA ^* is a mutant constructed similarly. (C)
FIG. 4 shows plasma resonance analysis of the interaction of purified recombinant KIF17 with purified recombinant mLin-10 or control BSA. (D) shows that recombinant KIF17 pulls down native mLin-10 into brain lysates. Wild ； KIF179
39-1038, Delta 3; KIF17 939-103
5, Delta 10; KIF939-1028, and KIFC2; KI
FC21-423 (E) shows immunoprecipitation of mLin-10 complex from brain.

FIG. 5 shows that the KIF17-mLin-10 complex binds to NMDAR2B-containing vesicles in a specific manner. (A)
FIG. 9 shows changes in developmental stages of expression of KIF17, mLin-10, mLin-2, mLin-7, NR2B and KIF5B. (B) shows the nycodenz float assay fraction. The bold curve shows the density of the fraction, and the thin curve shows the protein concentration of the fraction. Cox and vesicles show other membrane proteins with different peaks. (C) shows immunoprecipitation from brain lysate or vesicle fraction. (D) shows co-localization of KIF17 or NR2B on vesicles along microtubules. Vesicle size or microtubule thickness is enhanced by the optical limit of resolution. (E) shows KIF17 tail peptide block analysis. Bars indicate number of NR2B + vesicles. Error bars are S
EM (n = 3). The upper frame shows the concentration of KIF17 tail peptide.

FIG. 6 shows that KIF17 is expressed in dendrites of neurons in mLin1.
0, indicates co-localization with NR2B and NR1.
(A) shows coronal sections of cerebral cortex double stained with αKIF17 and αNR1 mAb. (B) shows coronal sections of the cerebral cortex treated with pepsin and double stained with αKIF17 and αNR2B (αGluRεNo59). (C) and (D) are αKI
Figure 2 shows cultured hippocampal neurons double stained with F17 and αmLin-10. The bar indicating the scale is 15 μm.

FIG. 7 shows visualization of NR2B transport by KIF17. (A) shows that movement of KIF17 transporter vesicles on the axon shows motility towards the plus end. The arrow is
Shows vesicles contacting, moving, and exiting on MT. (B)
Shows two time-laps of KIF17 transporter vesicle movement on microtubules
The e image is shown. (C) shows immunofluorescence detection of NR2B in vesicles moving on microtubules by KIF17. (D) KI on microtubules
FIG. 9 shows EM immune cell analysis of vesicles retaining F17. NR2B is detected with 10 nm gold particles. Scale bar is 1
00 nm. (E) shows a model of the NR2B transport mechanism.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 25/02 103 A61P 25/16 4B065 25/14 25/28 4C084 25/16 25/32 4H045 25/28 C07K 14/47 25/32 16/40 C07K 14/47 C12N 1/15 16/40 1/19 C12N 1/15 1/21 1/19 9/16 B 1/21 C12P 21/02 C 5/10 C12Q 1/02 9/16 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/02 C12P 21/08 G01N 33/15 C12N 15/00 ZNAA 33/50 A61K 37/02 // C12P 21/08 C12N 5/00 A F term (reference) 2G045 AA35 BB10 BB14 BB20 BB22 BB24 BB29 BB51 FA16 FA40 FB02 FB03 GC15 4B024 AA01 AA11 BA11 CA04 GA11 HA01 HA03 HA14 HA15 HA17 4B050 CC03 DD11 LL01 4B063 QA01 QA38 QA01 QA18 QA01 CA10 CA19 CA20 CC24 DA01 DA13 4B065 AA01X AA72X AA90X AA91Y AB01 AC14 BA01 CA24 CA44 4C084 AA02 AA06 AA07 AA13 BA01 BA08 BA22 BA23 CA17 CA18 CA23 CA28 CA53 NA14 ZA022 ZA152 ZA162 ZA212 ZA222 AA24A76 DAA

Claims (11)

[Claims] 1. A protein according to any one of the following (a) to (c): (A) a protein having the amino acid sequence of SEQ ID NO: 1; (b) an amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted and / or added, and mLin-10 Binds to NMDA receptor 2
A protein having an activity of transporting B (NR2B); and (c) an NMDA receptor 2B having an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 and binding to mLin-10. A protein having an activity of transporting (NR2B). 2. D encoding the protein according to claim 1.
NA. 3. Any one of the following DNAs. (A) a DNA having the nucleotide sequence of SEQ ID NO: 3; (b) a DNA having the nucleotide sequence of SEQ ID NO: 3 in which one or more bases have been deleted, substituted and / or added, and mLin-10 DNA encoding a protein having an activity of transporting NMDA receptor 2B (NR2B) by binding to DNA; (c) hybridizing with the DNA of (a) or (b) above under stringent conditions, and DNA encoding a protein having an activity of transporting NMDA receptor 2B (NR2B) by binding to -10. 4. A recombinant vector containing the DNA according to claim 2 or 3. A transformant having the recombinant DNA according to claim 4. 6. The transformant according to claim 5, which is cultured in a medium to produce the protein according to claim 1 in a culture,
2. The method of claim 1, comprising harvesting said protein from said culture.
2. The method for producing a protein according to item 1. 7. An antibody that recognizes the protein according to claim 1, or a fragment thereof. 8. The protein according to claim 1 and at least
A protein complex containing mLin-10. 9. A method for screening for a substance that specifically interacts with the protein, comprising using the protein according to claim 1 or the transformant according to claim 5. 10. A medicine comprising the protein according to claim 1 or the DNA according to claim 2 or 3 as an active ingredient. 11. The medicament according to claim 10, which is a medicament for treating and / or preventing a disease associated with abnormal nerve cell function.