JP2003286188A - Control agent for signal transduction through neurotrophin receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective means useful for controlling signal transduction through neurotrophin receptor p75 and useful for treating or preventing diseases caused by abnormality of the signal transduction through the neurotrophin receptor p75. <P>SOLUTION: A control agent for the signal transduction through p75NTR contains polypeptides having a sequence No.2 (refer to the specifications) and a sequence derived therefrom as effective components; contains an antibody specifically bonding to the polypeptide or its flagments as effective components; contains a nucleic acid for coding the polypeptide as effective component; contains an antisense nucleic acid of the nucleic acid as effective component. A medicinal composition for treating or preventing the diseases caused by abnormality of the signal transduction through p75NTR contains the control agent as an effective component. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a modulator capable of modulating signal transduction mediated by neurotrophin receptor p75; and a composition for treating or preventing a disease caused by an abnormality in the signal transduction. .

[0002]

BACKGROUND OF THE INVENTION Neurotrophins bind to two types of cell surface receptors, the Trk tyrosine kinase group and the p75 neurotrophin receptor (p75 ^NTR ) to maintain and differentiate neurons,
Mediates growth and apoptosis [Kaplan,
DR) et al., Curr. Opin. Neurobol., 10, 381-391 (200
0); Lee, FS et al., Curr. Opin. Neurobiol. 11,
281-286 (2001)].

[0003] In cells expressing both Trk and p75 ^NTR, p75 ^NTR is, T for neurotrophin
Adjusting the affinity of rk receptors [Hemstead (Hem
pstead, BL) et al., Nature, 350, 678-683 (1991; Barker, PA et al., Neuron, 13, 203-215 (1994)] and increasing ligand specificity [Beneditti (B
enedetti, M.) et al., Proc. Natl. Acad. Sci. USA, 90,
7859-7863 (1993); Bibel, M. et al., EMBO J.,
18, 616-622 (1999); Brennan et al., Nat.
Neurosci., 2, 699-705 (1999)] is known. On the other hand, in cells expressing p75 ^NTR alone, p75
^NTRs transmit their own signals. Also, Trk
Receptors transduce positive signals such as maintenance of survival and promotion of growth, but p75 ^NTR transduces both positive and negative signals.

However, the correlation between the various biological functions and p75 ^NTR signaling mechanisms that correspond to their extensive p75 ^NTR is at present, largely unknown.

Currently, means for treating or preventing neurodegenerative diseases such as Alzheimer's disease are being sought.

[0006] However, the treatment of central nervous system damage and neurodegenerative diseases is currently difficult because it is not fully understood why the damaged or degenerated adult central nervous system does not regenerate. is there.

[0007]

INDUSTRIAL APPLICABILITY The present invention is useful for regulation of neurotrophin receptor p75-mediated signal transduction, and treats or prevents diseases caused by abnormal neurotrophin receptor p75-mediated signal transduction. The purpose is to provide a useful means for doing so.
Specifically, the present invention relates to the neurotrophin receptor p7
It is an object of the present invention to provide a modulator of signal transduction mediated by the neurotrophin receptor p75, which is useful for regulation of signal transduction mediated by 5. Further, the present invention provides treatment or prevention of diseases caused by abnormal neurotrophin receptor p75-mediated signal transduction, which are useful for treatment or prevention of diseases such as neurodegenerative diseases, central nerve injury, neurogenic tumors and the like. An object is to provide a pharmaceutical composition for use.

[0008]

The summary of the present invention is as follows.
[1] The following amino acid sequences (a) to (f): (a) the amino acid sequence shown by SEQ ID NO: 2, (b) the amino acid sequence having at least 97% sequence identity with said SEQ ID NO: 2, ( c) substitution of at least one amino acid residue,
An amino acid sequence different from said SEQ ID NO: 2 through deletion, addition or insertion, (d) in (c) above, amino acid numbers: 4, 5, 6, 7, 10, 10 in said SEQ ID NO: 2
Amino acid sequence in which 2, 13, 14 and 15 are conserved, (e) the sequence of any one of (a) to (d) through at least one amino acid residue that imparts physiologically equivalent physicochemical properties An amino acid sequence different from the above, and (f) in the living body or in a physicochemical environment equivalent to the living body,
To an amino acid sequence capable of supplying a polypeptide having a physiologically equivalent three-dimensional structure to the polypeptide consisting of the amino acid sequence of (e), and a neurotro Fin receptor p75
(Gene bank accession number: NM — 002507), a regulator of signal transduction through the neurotrophin receptor p75, comprising a polypeptide having an activity of phosphorylating (2) as described in [1] above. Neurotrophin receptor p comprising as an active ingredient an antibody or a fragment thereof that specifically binds to a polypeptide
[3] A neurotrophin receptor p75 containing a nucleic acid encoding the polypeptide according to [1] above as an active ingredient.
[4] The nucleic acid is a base sequence represented by the following (A) to (F): (A) SEQ ID NO: 1, and (B) the nucleotide sequence via degeneracy with SEQ ID NO: 1. Different nucleotide sequences, (C) a nucleotide sequence having a sequence identity of at least 97% with said SEQ ID NO: 1, and the encoded polypeptide exhibits p75 phosphorylation ability, (D) said The base numbers: 336 to 347, the base numbers: 354 to 356, and the base numbers: 360 to 371 in SEQ ID NO: 1 are conserved, and the SEQ ID NO: is obtained through substitution, deletion, addition or insertion of at least one base. A polypeptide that is different from 1 and is capable of hybridizing under stringent conditions to the complementary strand of the nucleic acid consisting of the nucleotide sequence (E) SEQ ID NO: 1, which has a phosphorylation ability of p75. The base sequence of a nucleic acid, which is encoded The encoded polypeptide is different from SEQ ID NO: 1 through the nucleotide sequence which exhibits phosphorylation ability of p75, and (F) nucleic acid polymorphism, and the encoded polypeptide is phosphorylated of p75. A nucleic acid containing a base sequence selected from the group consisting of a base sequence exhibiting a function, and the nucleic acid described in [3] or [4] above, [5] [6] A regulator of signal transduction mediated by neurotrophin receptor p75, comprising an antisense nucleic acid having a sequence complementary to as an active ingredient, [6] any one of [1] to [5] above. [7] Neurotrophin, which comprises as an active ingredient the regulator of the present invention, for treating or preventing a disease caused by an abnormality in signal transduction through the neurotrophin receptor p75, [7] Neurotrophin Receptor diseases caused by abnormal signaling through p75 is, neurodegenerative disease, a disease selected from the group consisting of neurogenic tumors, the [6] pharmaceutical composition for treating or preventing according,
Regarding

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to protein kinase A.
The splice variant of the catalytic subunit of (PKA) is a neurotrophin receptor p75 (hereinafter, p75).
(Also referred to as ^NTR ), specifically p
Our findings that phosphorylate 75 ^NTR and binding of ligand (NGF) to p75 ^NTR resulted in cAM
It is based on the findings of the present inventors that P accumulates and PKA is activated, and the specific membrane localization of p75 ^NTR and the downstream signal are regulated.

With respect to the PKA, the inactive PKA holoenzyme exists as a heterotetramer consisting of two regulatory (R) subunits and two catalytic (C) subunits. When 4 molecules of cAMP bind to the R subunit dimer and 2 molecules bind to each subunit with positive cooperativity, P
Activation of KA occurs. The C subunit consists of two isoforms, Cα and Cβ, which have been characterized in various species. The Cα isoform is ubiquitously expressed, while Cβ is highly expressed in the brain. In bovine, each of the Cβ splice variants is 2
One has been identified, and three have been identified in mice [Wiemann, SJ Biol. Chem. 15, 51.
40-5146 (1991); Guthrie, CR et al., J. Bi
ol. Chem., (1997)]. On the other hand, regarding the human Cβ gene, the existence of at least six kinds of gene products named Cβ1, Cβ2, Cβ3, Cβ4, Cβ4ab and Cβ4abc has been suggested [Orstavik (Orstavi
k, S.) et al., J. Biochem., 268, 5066-5073, (2001)].

The p75 ^NTR is a neurotrophin,
For example, it is a low-affinity receptor for NGF (nerve growth factor) and forms a receptor complex of NGF in cooperation with the Trk tyrosine kinase group.

The biological function of p75 ^NTR is as follows:
For example, induction of cell death, enhancement of axon growth, action on Schwann cell migration, regulation of synaptic transmission, regulation of sensory neuron function, regulation of calcium current and the like can be mentioned.

The p75 ^NTR is also localized in the raft and soluble fractions of cerebellar neurons. Lipid rafts are lipid microdomains of eukaryotic cell membranes that are rich in cholesterol and sphingolipids and are thought to function in cell signaling in neurons by concentrating or sequestering specific molecules into a distinct lipid environment.

Signal transduction molecules downstream of p75 ^NTR include, for example, JNK-p53-Bax, Rho, Ra.
c, ceramide, NF-κB, MAPK, PI3K and the like.

The regulator of signal transduction mediated by p75 ^NTR of the present invention contains a substance involved in phosphorylation of p75 ^NTR , and specifically, a regulator containing a polypeptide of PKA catalytic subunit. (A regulator of aspect 1), a regulator containing an antibody or an antibody fragment against the polypeptide (a regulator of aspect 2), a regulator containing a nucleic acid encoding the polypeptide (a regulator of aspect 3), Regulator containing antisense nucleic acid to nucleic acid (Aspect 4
Regulator). According to modifiers of the present invention, the can modulate signaling via p75 ^NTR, which makes it possible to act on the signal transduction downstream of the p75 ^NTR.

The polypeptide used in the modulator of aspect 1 contains the amino acid sequence shown in SEQ ID NO: 2 [amino acid sequence (a)] or a sequence similar to the amino acid sequence shown in SEQ ID NO: 2, In addition, one of the major features is that it has an activity of phosphorylating p75 ^NTR (Gene Bank Accession No. NM_002507). According to the regulator of Embodiment 1 of the present invention, since it contains the above-mentioned polypeptide, it is possible to cause an interaction with p75 ^NTR, and specifically, consensus PKA phosphorylation in the linker region near the p75 ^NTR membrane. Phosphorylates the site in vitro and binds the ligand (NGF) to p75 ^NTR to c
AMP accumulates, which activates PKA and results in p7
5 It exerts an excellent effect of regulating the specific membrane localization and downstream signal of ^NTR .

The amino acid sequence of (a) above (SEQ ID NO:
2) is the sequence of the splice variant of the catalytic subunit of PKA. Such a splice variant was obtained as a p75 ^NTR interacting protein from a human fetal brain cDNA library using the yeast two-hybrid system.

The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is the human Cβ4ab,
Human Cβ4ab mRNA is expressed in the brain.

The "similar sequence of the amino acid sequence shown in SEQ ID NO: 2" may be a sequence of a polypeptide exhibiting the activity of phosphorylating p75 ^NTR , for example, (b)
An amino acid sequence having a sequence identity of at least 97% with said SEQ ID NO: 2, (c) an amino acid sequence different from said SEQ ID NO: 2 through substitution, deletion, addition or insertion of at least one amino acid residue, (D) In the above (c), amino acid numbers: 4, 5, 6, 7, 1 in the SEQ ID NO: 2
An amino acid sequence in which 0, 12, 13, 14 and 15 are conserved,
(E) an amino acid sequence different from any of the sequences of (a) to (d) via at least one amino acid residue that imparts physiologically equivalent physicochemical properties, and (f) in vivo or An amino acid sequence that can supply a polypeptide that can have a physiologically similar three-dimensional structure to the polypeptide consisting of the amino acid sequences (a) to (e) under the same physicochemical environment as in the living body, and the like. To be

Whether or not the polypeptide exhibits the activity of phosphorylating p75 ^NTR is determined by, for example, obtaining a polypeptide having a desired amino acid sequence,
The polypeptide can be evaluated by subjecting it to the method for measuring phosphorylation activity in Example 3 described later and measuring the phosphorylation activity.

The polypeptide having a desired amino acid sequence is, for example, a conventional method for producing a polypeptide, specifically, the following steps i) to v): i) Designing a nucleic acid encoding the desired amino acid sequence Step ii) ligating the nucleic acid obtained in i) to an appropriate expression vector to obtain a recombinant vector, iii) introducing the recombinant vector obtained in ii) into a host cell, and transforming Obtaining a cell or a transfected cell, iv) culturing the transformed cell or the transfected cell obtained in iii) above under conditions suitable for the expression of the polypeptide, and then in the transformed cell or the transfected cell. Expressing or producing the desired polypeptide, or secreting the desired polypeptide into the culture medium, v) collecting the cells or the culture medium in which the polypeptide was expressed in iv) above, In quality purification methods, obtained by a method or the like including a process of isolating the polypeptide.

In the step i), the nucleic acid encoding the desired amino acid sequence is obtained, for example, by a conventional nucleic acid synthesis method to obtain a primer pair having a sequence capable of supplying the desired sequence. A nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2 (SEQ ID NO:
It can be prepared by PCR using 1) and.

In the step ii), the expression vector is a promoter suitable for gene expression (for example, l
ac promoter, tac promoter, trc promoter, trp promoter, CMV promoter, S
V40 early promoter, etc.), a vector having a transcriptional control sequence such as an enhancer, etc., and can be selected from conventional expression vectors depending on the host to be used. The conventional expression vector is not particularly limited, but for example, p
LP, pDNR, pcDNA, pCMV, pGEX, p
ET, pQE, pKC, pDR, pMAL, pBT, p
RIT, pKK, etc. are mentioned. In addition, using a vector having a sequence corresponding to another polypeptide (glutathione-S-transferase etc.) or tag sequence (histidine tag) of the target polypeptide that can facilitate purification, specifically, pGEX-5X or the like is used. Good. The nucleic acid in step i) may be linked to the transcription control sequence and the obtained nucleic acid may be supported on liposomes, metal particles or positively charged polymer and used in step iii) described later.

Then, in the step iii), the host cells are Escherichia coli (HB101, JM109, DH5α).
Etc.), yeast cells (Saccharomyces cerevisiae etc.), animal cells (COS cells, CHO cells, L cells, 3T3 cells, Vero cells, HeLa cells etc.), insect cells (sf
9 etc.). The method of introducing the vector into the host cell is appropriately selected according to the vector to be used, and examples thereof include the calcium phosphate method, the DEAE-dextran method, the electric pulse method, and the particle bombardment method.

In step iv), pGEX-5X is used as an expression vector, and E. coli DH5 α is used as a host.
When used, the transformant is grown to an OD ₆₀₀ of 0.6 and 1 mM isopropyl-1-thio-β-D-
Galactopyranoside (IPTG) can be added to the culture to induce peptide synthesis, and then the cells can be grown at 25 ° C. for an additional 16 hours to obtain the GST fusion polypeptide of the desired polypeptide. The target polypeptide is either expressed intracellularly or extracellularly (in culture) depending on the type of vector used and the type of host cell.
Secreted by.

In step v), the cell or culture is subjected to a conventional protein purification method such as salting out, ion exchange column chromatography, adsorption column chromatography, affinity column chromatography to obtain the desired polypeptide. Obtainable.

When using the above-mentioned pGEX-5X, 1) a cell extract is prepared from the obtained culture, and 2) the cell extract is subjected to glutathione affinity chromatography to obtain a GST fusion protein of p75ICD,
3) Incubating the obtained protein with Factor Xa overnight at 4 ° C. to remove the GST moiety, 4) obtaining the product with ion exchange H on a DEAE cellulose column.
The target polypeptide can be obtained by subjecting to PLC.

As a method for measuring phosphorylation activity, for example,
I) a polypeptide having the desired amino acid sequence
mM HEPES (pH 7.5) and 10 mM MgCl
₂ and 5 mM dithiothreitol and 100 mM N
aCl and 1 mM [γ- ³² P] ATP (0.5 Ci /
50 μm by adding to a PKA reaction buffer containing
Incubate for 30 minutes at 30 ° C. with 50 U of PKA catalytic subunit [Calbiochem] for a total volume of 1; II) The product obtained in step I) was subjected to SDS polyacrylamide electrophoresis. And a step of III) measuring the radioactivity of a band corresponding to the polypeptide on the SDS polyacrylamide gel electrophoresis obtained in step II). In addition, histone proteins and the like can be used as a positive control.

In (b) above, the sequence identity with SEQ ID NO: 2 is at least 97%, specifically 98% or more, preferably 99%, based on an algorithm for obtaining optimal alignment and homology. % Or more, more preferably 100% or more. Examples of the algorithm include local homology algorithm [Add. APL. Math., 2, 482 (1981)] by Smith et al., Homology alignment algorithm [J. Mol. Biol by Needleman] et al. ., 48, 443 (1970)], Pearlson (P
homology search method from Earson et al. [Proc. Natl. Acad. Sci. US
A, 85, 2444 (1988)], more specifically, dynamic programming method, gap penalty method,
Smith-Waterman algorithm, Good-Kanehisa algorithm, BLAST algorithm, FASTA algorithm, etc. are mentioned. In addition, such sequence identities can be determined, for example, by a conventional analysis software or program based on the above-mentioned algorithm, for example, BLAST (http://www.nlm.nih.gov/BL).
(Generally available on the website shown in AST) etc. In the present specification, hereinafter, the sequence identity based on the BLAST algorithm is shown, but the sequence identity calculated based on another algorithm may be included within the range under the BLAST algorithm. . In the present invention, any polypeptide having an amino acid sequence having the above sequence identity may be one that exhibits phosphorylation activity by the above-mentioned phosphorylation activity measurement method.

The amino acid sequence of (c) may be a sequence in which amino acid substitutions, deletions, additions or insertions have been artificially introduced by a conventional site-directed mutagenesis method or the like. It may be an existing sequence. In the present specification, the number of “substitution, deletion, addition or insertion” of the polypeptide is at least one amino acid residue, if the polypeptide exhibits phosphorylation activity by the above phosphorylation activity measurement method, Specifically, the number is one or more or several or more. More preferably, in the amino acid sequence of (c) above, the amino acid sequence in which the amino acid numbers: 4, 5, 6, 7, 10, 12, 13, 14 and 15 in the SEQ ID NO: 2 are conserved [amino acid sequence (d )].

In the above (e), an amino acid that imparts physiologically equivalent physicochemical properties means that the expression of the physiological activity of the polypeptide, that is, the activity of phosphorylating p75 ^NTR , is maintained, and An amino acid that can exhibit the same physicochemical properties with respect to shape characteristics, hydrophobicity, electric charge, pK, etc. in the three-dimensional structure. For example, the side chain of the amino acid of the amino acid sequence shown in SEQ ID NO: 2 is represented by, for example, Amino acid analogs obtained by substituting other side chains showing bioisosterism, groups of amino acids having the same physicochemical properties, for example, a group consisting of glycine and alanine; valine, isoleucine and leucine Group consisting of: aspartic acid, glutamic acid, asparagine and glutamine, serine and threonine Ranaru group; lysine and group consisting of arginine; within any of the groups of the group consisting of and phenylalanine and tyrosine, other amino acids in the same group can be mentioned. The number of amino acid residue changes should be at least 1.
It is an amino acid residue, specifically, one or more or several or more.

In the above (f), the physicochemical environment equivalent to that in the living body means that the conditions such as pH, ion concentration, temperature, etc. are the same as those in the living body. Polypeptides differ from each other at the primary structural level of their amino acid sequences,
In a living body or a physicochemical environment equivalent to that in the living body, a similar three-dimensional structure may be exhibited. In the present invention,
A polypeptide that exhibits a similar three-dimensional structure in the above environment and exhibits phosphorylation activity by the method for measuring phosphorylation activity is also included. Therefore, the amino acid sequence of (f) means an amino acid sequence capable of supplying such a polypeptide.

The polypeptide used in the present invention may have any length as long as it includes any of the amino acid sequences (a) to (f) described above, and at least 300 residues, preferably 300 to. 340 residues, more preferably,
It is preferably 300 to 320 residues. In addition, it is desirable that the above-mentioned polypeptide contains a region of positions 47 to 346 in the amino acid sequence shown in SEQ ID NO: 2 or a portion corresponding to the region. "47-34
The "portion corresponding to the region at position 6" means a derivative sequence which exhibits phosphorylation activity equivalent to that of the region at positions 47 to 346 in the amino acid sequence shown in SEQ ID NO: 2.

The function of the PKA catalytic subunit for p75 ^NTR can be regulated by an antibody or fragment thereof that specifically binds to the polypeptide. Therefore, the modulator of the present invention may be a modulator containing the antibody of the above-mentioned polypeptide or a fragment thereof (modulator of aspect 2).

Since the modulator of the second aspect of the present invention contains the antibody or a fragment thereof, it can inhibit the function of the PKA catalytic subunit for p75 ^NTR ,
It exerts an excellent effect that it can regulate the signal transduction mediated by the neurotrophin receptor p75.

The antibody or fragment thereof used in the present invention is
An antiserum can be obtained by immunizing animals such as rabbits, rats and chickens with the above-mentioned polypeptide together with an adjuvant. Also, the method of Galfre et al.
fre, G. et al., Nature, Volume 266, Volumes 550-552
Page (1977)], it can also be obtained as a monoclonal antibody. Furthermore, the obtained antibody is purified and then treated with peptidase or the like to obtain an antibody fragment.

The antibody or fragment thereof used in the present invention is
Either a polyclonal antibody or a monoclonal antibody may be used. Further, it may be an antibody or an antibody derivative modified by a known technique, such as a Fab fragment or a single chain antibody. Furthermore, the antibody or fragment thereof used in the present invention may be a humanized antibody.

According to the nucleic acid encoding the above-mentioned polypeptide, the PKA for p75 ^NTR is expressed by expressing the above-mentioned polypeptide or expressing or complementing the function of the above-mentioned polypeptide in the target substance, specifically, in vivo. It exerts an excellent effect of expressing the function of the catalytic subunit and being able to regulate the signal transduction mediated by the neurotrophin receptor p75. Therefore, the regulatory agent of the present invention may be a regulatory agent (a regulatory agent of aspect 3) containing a nucleic acid encoding the above-mentioned polypeptide.

Specific examples of the nucleic acid used in the present invention include the following (A) to (F): (A) the nucleotide sequence shown in SEQ ID NO: 1 and (B) the nucleotide sequence degenerate with the SEQ ID NO: 1. (C) a nucleotide sequence having a sequence identity of at least 97% with said SEQ ID NO: 1, and the encoded polypeptide exhibits phosphorylation ability of p75 (D) ) The nucleotide numbers: 336 to 347, the nucleotide numbers: 354 to 356, and the nucleotide numbers: 360 to 371 in the SEQ ID NO: 1 are conserved, and the sequence is obtained through substitution, deletion, addition or insertion of at least one nucleotide. A hybridized to a complementary strand of a nucleic acid consisting of (E) SEQ ID NO: 1, which is different from SEQ ID NO: 1 and in which the encoded polypeptide exhibits a phosphorylation ability of p75, under stringent conditions The nucleotide sequence of a possible nucleic acid, Encoded polypeptide, nucleotide sequence is one that exhibits the ability to phosphorylate p75, through (F) a nucleic acid polymorphisms,
A nucleic acid containing a base sequence selected from the group consisting of a base sequence different from SEQ ID NO: 1 and having an encoded polypeptide exhibiting p75 phosphorylation ability. In the present invention, the 327th to 1st positions of SEQ ID NO: 1 which is the coding region of the polypeptide of the present invention.
A nucleic acid containing a base sequence of the region consisting of position 388 or a derivative sequence thereof can also be used.

Since the regulator of the third aspect of the present invention contains the above-mentioned nucleic acid, it is excellent in that the above-mentioned polypeptide can be efficiently supplied in an object to be introduced, specifically, in vivo. Exert the effect. In addition, according to the modulator of the third aspect of the present invention, the above-mentioned polypeptide can be expressed or the function of the polypeptide can be expressed or complemented, thereby regulating the signal transduction mediated by neurotrophin receptor p75. It has the excellent effect of being able to.

In (A) above, the base sequence of SEQ ID NO: 1 is one of the sequences corresponding to the amino acid sequence of SEQ ID NO: 2. A plurality of codons may exist as corresponding codons in amino acids. Therefore, a nucleic acid containing a base sequence different from SEQ ID NO: 1 due to degeneracy can also be used in the present invention.

In (C) above, the sequence identity with SEQ ID NO: 1 may be such that the encoded polypeptide exhibits phosphorylation activity according to the above phosphorylation activity measurement method, and is at least 97%, It is preferably 98% or more, more preferably 99% or more.

The amino acid sequence of the above (D) is artificially substituted for a base by a conventional site-directed mutagenesis method or the like,
It may be a sequence introduced with a deletion, addition or insertion,
It may be a naturally occurring sequence. In the present specification, the encoded polypeptide is one that exhibits phosphorylation activity according to the method for measuring phosphorylation activity,
The number of “substitutions, deletions, additions or insertions” is at least one base, and specifically, one or more or several or more.

In the above (E), stringent conditions may be such that the polypeptide encoded by the hybridized nucleic acid exhibits phosphorylation activity by the above-mentioned phosphorylation activity measurement method, for example, molecular weight.・ Cloning: A laboratory manual, second
Ed., Conditions described in pages 9.52 to 9.55 (1989), and specifically, specifically, for example, 0.
5% SDS, 0.1% bovine serum albumin (BS
A), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400, 0.01% 6 × SSC containing denatured salmon sperm DNA (1 × SSC is 0.15M NaCl,
Incubation at 50 ° C. in 0.015 M sodium citrate, pH 7.0).

In the above (F), the nucleic acid polymorphism may be any one as long as the encoded polypeptide exhibits p75 phosphorylation ability.

The length of the nucleic acid used in the present invention is
The length may be at least 900 nucleotides, preferably 900 to 1020 nucleotides, more preferably 9 nucleotides, as long as it includes any one of the base sequences (A) to (F).
It is preferably from 00 to 960 nucleotides. It is desirable that the nucleic acid contains a region consisting of the sequences at positions 465 to 1364 in the base sequence shown in SEQ ID NO: 1 or a portion corresponding to the region. In addition, "4
The portion corresponding to the region consisting of the sequences at positions 65 to 1364 "
Means a polypeptide encoded by a region consisting of the sequences at positions 465 to 1364 shown in SEQ ID NO: 1, for example,
47-3 in the amino acid sequence shown in SEQ ID NO: 2
It means a derivative sequence that encodes a polypeptide that expresses phosphorylation activity equivalent to that of the amino acid at position 46.

The function of the PKA catalytic subunit for p75 ^NTR is regulated by the antisense nucleic acid for the nucleic acid, for example, by inhibiting the expression of the polypeptide from any of the nucleic acids (A) to (F) above. It is possible to Therefore, the regulator of the present invention may be a regulator containing an antisense nucleic acid to the nucleic acid (the regulator of the embodiment 4).

According to the fourth regulator of the present invention, since it contains the antisense nucleic acid, it inhibits the transcription / expression from the nucleic acid consisting of SEQ ID NO: 1 and thus mediates the neurotrophin receptor p75. It exerts an excellent effect of being able to regulate signal transduction. Furthermore, the fourth modulator of the present invention is expected to be applied to the treatment or prevention of a disease caused by an abnormality in signal transduction mediated by neurotrophin receptor p75.

The antisense nucleic acid used in the present invention is
It can be prepared by a conventional method. Moreover, the length of the antisense nucleic acid is not particularly limited, and is 12 to 10
Desirably, it has 0 nucleotides, preferably 15 to 50 nucleotides, and more preferably 15 to 30 nucleotides. The antisense nucleic acid used in the present invention is preferably an antisense nucleic acid capable of specifically inhibiting, suppressing or reducing the transcription from the nucleic acid and the expression of the polypeptide. For example, a sequence that cannot be interacted with a nucleic acid that is not related to the nucleic acid used in the present invention from a commonly available database, such as the above-mentioned “unrelated to the nucleic acid used in the present invention”. A sequence having a sequence identity with "nucleic acid" of 10% or less, preferably 5% or less, more preferably 0% may be designed.

The modulators of the present invention include the polypeptides described above,
The antibody or fragment thereof, the nucleic acid and the antisense nucleus
Contains one active ingredient selected from the group consisting of acids
There is one major feature in this. The regulator of the present invention is
The polypeptide, the antibody or fragment thereof, the nucleic acid or
Contains the above antisense nucleic acid, so p75
^NTRIt is expected that the signal transduction via the cell is regulated.

When a significant change occurs in the signal recognized to occur through p75 ^{NTR in} cells, for example, inactivation of Rho, activation of Rac, NF
The case where a significant change occurs in activation of _−k B, activation of JNK, etc. can be evaluated as an index.

The content of the active ingredient in the regulator of the present invention may be an amount sufficient to regulate the signal transduction mediated by p75 ^NTR . For example, in the case of a polypeptide,
0.01-1000 μg, preferably 0.1-10 μg
g, more preferably 1 to 10 μg, and in the case of an antibody or antibody fragment, 0.01 to 1000 μg, preferably 0.1 to 10 μg, more preferably 1 to 10 μg.
g, 0.01 to 100 in the case of nucleic acid or antisense nucleic acid
The amount may be 0 μg, preferably 0.1 to 10 μg, and more preferably 1 to 10 μg.

Such regulators are auxiliaries suitable for introduction into a living organism, as long as they do not interfere with the regulation of signal transduction mediated by p75 ^NTR ; the polypeptide, the antibody or fragment thereof, the nucleic acid, the anti-antibody. It may further contain a stabilizer for the sense nucleic acid.

According to the present invention, there is provided a pharmaceutical composition for treating or preventing a disease caused by an abnormality in p75 ^NTR- mediated signal transduction, which comprises the above-mentioned modulator as an active ingredient.

Since the therapeutic or prophylactic pharmaceutical composition of the present invention contains the above-mentioned regulator as an active ingredient, p75
^In diseases caused by abnormal signal transduction through ^NTR , it is expected that the signal transduction can be regulated to normal state, thereby treating or reducing the symptoms.

The content of the regulator in the therapeutic or prophylactic pharmaceutical composition of the present invention is appropriately selected depending on the disease; the individual, organ, local site, tissue to be administered; age of the individual to be administered, etc. The amount of the polypeptide, antibody or fragment thereof, nucleic acid or antisense nucleic acid which can be selected is p
The amount may be sufficient to regulate the signal transduction through 75 ^NTR, and in the case of a polypeptide, for example, 0.01 to
1000 μg, preferably 0.1 to 10 μg, more preferably 1 to 10 μg, and 0.01 to 1000 μg, preferably 0.1 in the case of an antibody or antibody fragment.
10 to 10 μg, more preferably 1 to 10 μg, and 0.01 to 1000 μg, preferably 0.1 to 10 μg, and more preferably 1 to 10 in the case of nucleic acid or antisense nucleic acid.
It may be μg.

Examples of the above-mentioned "disease caused by abnormal signal transduction via p75 ^NTR " include neurodegenerative diseases and neurogenic tumors.

It is considered that the damaged central nervous system of the adult does not regenerate due to the presence of a nerve regeneration inhibitor in the adult central nervous system. It has been proved that the p75 receptor plays an important role when a nerve regeneration inhibitor transmits its signal to nerve cells. According to the therapeutic or prophylactic pharmaceutical composition of the present invention, by promoting or suppressing phosphorylation of p75, its distribution in the cell membrane can be changed and signal transduction can be controlled. Therefore, the therapeutic or prophylactic pharmaceutical composition of the present invention may be capable of suppressing the effects of nerve regeneration inhibitors, and as a result,
It will be possible to regenerate the central nervous system of an injured adult,
It can be expected to be effective as a therapeutic drug for nerve palsy due to spinal cord injury, cerebral trauma, cerebral infarction, and cerebral hemorrhage.

The therapeutic or prophylactic pharmaceutical composition of the present invention may be an agent obtained by supporting the active ingredient on a carrier capable of stably maintaining the active ingredient. Specifically, for example, the active ingredient may be held in a pharmaceutically acceptable carrier that can facilitate introduction into an organism such as an individual, organ, local site, or tissue to be administered.

The therapeutic or prophylactic pharmaceutical composition of the present invention further contains other auxiliaries depending on the type of active ingredient, disease, individual to be administered, organ, local site and tissue. Good. Specifically, it is a pharmaceutically acceptable substance that exhibits the property of suppressing the degradation of a polypeptide, an antibody or a fragment thereof, a nucleic acid or an antisense nucleic acid before reaching the site where the effect of the active ingredient is expressed. Auxiliary agents, excipients, binders, stabilizers, buffers, solubilizing agents, isotonic agents and the like may be contained.

When the therapeutic or prophylactic pharmaceutical composition of the present invention contains a polypeptide as an active ingredient, it has a therapeutic effect on the above-mentioned diseases, particularly against neurodegenerative diseases and neurogenic tumors. Be expected. Moreover, when an antibody or a fragment thereof is contained as an active ingredient, a therapeutic effect is expected among the above-mentioned diseases, particularly against neurodegenerative diseases and neurogenic tumors. Furthermore, when a nucleic acid is contained as an active ingredient, a therapeutic effect is expected among the above diseases, particularly against neurodegenerative diseases and neurogenic tumors. In addition, when an antisense nucleic acid is contained as an active ingredient, a therapeutic effect is expected especially for neurodegenerative diseases and neurogenic tumors among the above diseases.

The dosage form and dose of the therapeutic or prophylactic pharmaceutical composition of the present invention are as follows: type of active ingredient, disease, individual to be administered, organ, local site, tissue; age and weight of individual to be administered. It is appropriately selected according to the above. Examples of the administration form include local administration, subcutaneous injection, intramuscular injection, intravenous injection, intrathecal administration and the like. When the active ingredient is a polypeptide, the dose of the active ingredient is 0.1 μm for an adult from the viewpoint of obtaining an appropriate effect.
g or more, preferably 0.2 μg or more, more preferably
0.3 μg or more and 10 μg or less, preferably 7 μg
The amount is preferably 5 μg or less, more preferably 3 times a day. further,
When the active ingredient is the antibody or a fragment thereof, the dose of the active ingredient is 0.1 μg or more, preferably 0.2 μg or more, and more preferably 0.3 μg for an adult from the viewpoint of obtaining an appropriate effect. It is desirable that the dose is not less than μg and not more than 10 μg, preferably not more than 7 μg, more preferably not more than 5 μg, and it is desirable to administer three times a day. When the active ingredient is a nucleic acid or an antisense nucleic acid, the dose of the active ingredient is 0.1 μg or more, preferably 0.2 μg for an adult from the viewpoint of obtaining an appropriate effect.
g or more, more preferably 0.3 μg or more, 10 μg
Or less, preferably 7 μg or less, more preferably 5 μg
It is preferably g or less, and it is desirable to administer 3 times a day.

When the active ingredient is a polypeptide, the polypeptide may be subjected to various modifications suitable for introduction into a living body.

When the active ingredient is a nucleic acid or an antisense nucleic acid, it is carried on a carrier such as a liposome and then introduced into a living body by a conventional method used for gene therapy or the like.
The therapeutic or prophylactic pharmaceutical composition of the present invention may be administered.

[0065]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to the examples. The operations and conditions in this embodiment are
Unless otherwise noted, for example, Molecular Cloning Laboratory Manual 3rd Edition [Sambrook et al., Molecular Cloning: A Laboratory M].
anual 3rd ed., Cold Spring Harbor Laboratory issued,
(2001)], Culture of Animal Cells a Manual of Basic Technique, 2nd Edition [Freshney, R. Ian] et al., Culture of animal
cells A manual of basic technique, 2nd ed., Alan
R. Liss. Inc., 66-84 (1987)] and the like.

Example 1 The classical yeast two-hybrid system may not be suitable for identifying membrane-bound proteins or proteins that are not localized in the nucleus.

Therefore, in a more natural environment, p75
To identify proteins that interact with the ^NTR intracellular domain (p75ICD), Son of seven
Yeast tw based on the less recruitment system
An o-hybrid assay was performed.

Yeast Son of sevenless
(Sos) recruitmenttwo-hybr
id system, Aronheim, A
[Mol. Cell. Biol., 17, 3094-3102, (1997)], custom two-hybr manufactured by Stratagene.
It was carried out by referring to the protocol attached to the id cDNA library.

In temperature-sensitive mutant yeast, the protein partner is myristylated on the membrane and bait is cytosolic So.
fused to the s protein. When an interaction occurs between the protein encoded by the partner and the protein encoded by bait, S in mutant yeast
It causes the translocation of os to the membrane and the activation of the Ras-related survival maintenance pathway. The protein encoded by bait does not activate the Ras signal transduction system by itself in yeast.

Bait was the entire cytoplasmic domain of human p75 ^NTR fused in frame to human Sos protein.

By PCR, human p75ICD cDNA
A was prepared. The obtained cDNA is used as the vector pSos.
To obtain a recombinant vector. Saccharomyces
Selviche (Saccharomyces cerevisiae) cdc25
-H strain was cloned with the above recombinant vector and human fetal brain-derived cD
Library of NA fragments [Stratagene by oligo (dT) primer method prepared from poly (A) ⁺ RNA
(Stratagene) custom two-hybrid cd
NA library] and the vector pMyr fused thereto. About 5 × 10 ⁶ transformants were screened and positive clones were isolated from temperature insensitive colonies. Extraction of plasmid from surviving colonies
Amplified in E. coli for sequence analysis.

Next, the specificity of the interaction between bait and the target protein was verified by cotransformation. MAFB
Since proteins can bind to each other, MA
FB was used as a positive control. Unrelated bait (Col
13) and target (Lamin C) were used as negative controls.

As a result, a human fetal brain cDNA library was screened to obtain 13 candidate clones. One of them encoded a splice variant of the β-catalytic subunit of cAMP-dependent protein kinase (PKACβ4). To assess specificity, irrelevant bait (Sos-Coll) or native
Yeast cells containing bait (Sos-p75ICD) were treated with PK
CDNA containing the open reading frame of ACβ4
Transformed with A, grown at 25 ° C, replicated on galactose and glucose plates and incubated at 37 ° C. The results are shown in Fig. 1.

As shown in FIG. 1, suppression of the cdc25-H temperature-sensitive phenotype results in Sos on galactose plates.
It can be seen that this occurs only in cells expressing the p75ICD fusion protein and PKACβ4. These results are
PKACβ4 is a yeast Sonof sevenless
It shows to interact specifically with p75ICD in the recruitment two-hybrid system.

Example 2 (1) Northern Blot Analysis In order to examine the functional role of PKA, the tissue distribution of PKACβ4ab was examined by conventional Northern blot analysis.

To specifically detect PKACβ4ab mRNA, exons 1-4 of PKACβ4ab, 1
A 370 bp fragment corresponding to -a and 1-b was used.

PCR was carried out using the cDNA clone as a template, and then the pGEM-T vector [Promega (Prome
ga)) to obtain a plasmid for preparing a cDNA probe for Northern blot analysis.

In order to prepare a PKACβ4ab-specific fragment, the following nucleotide sequences: 5'-CCCTGCCATTTTGAGTTGTTC-3 '(SEQ ID NO: 3) and 5'-TATTTCTGAAGAGGAGCAAGC-3' (SEQ ID NO: 4) were used. The following PCR primers were used.

The PKACβ4ab-specific fragment thus obtained was
Using the DNA labeling kit (Takara Shuzo), [α-
Labeled with ³² P] dCTP, then the labeled fragment was isolated on a Sephadex G-75 column.

The labeled fragment thus obtained and the human MTN blot (manufactured by Clontech) were used to prepare a hybridization buffer solution [composition: 6 × SSC (1 × SSC:
16.7 mM sodium chloride, 16.7 mM sodium citrate
[pH 7.0]), 0.5% SDS, 100 μg / ml herring sperm DNA and 5 × Denhardt's solution] at 68 ° C. After overnight hybridization, the blots obtained were stained with 1 × SSC and 0.1%.
It was washed twice at 68 ° C. in a solution containing SDS. Then, the obtained blot was applied to X-Omat AR [Eastman Kodak Company] at -80 ° C.
Exposed for 48 hours.

As a result, by Northern blot analysis of human-derived tissues, it was found that, as shown in FIG. 2, there is an mRNA species of about 4.6 kb showing a high expression level in the brain. However, in other tissues tested, PKACβ
No 4ab mRNA expression was seen. These results revealed the specific localization of PKACβ4ab in the brain.

(2) Fluorescence Microscopic Analysis The localization of PKACβ variants in cells was evaluated using an expression vector incorporating the GFP fusion protein.

That is, using the pEGFP-N1 expression plasmid (manufactured by Clontech), a human PKACβ1 construct having an enhanced green fluorescent protein (EGFP) tag at the amino terminus and PKACβ4ab.
And construct. 293T using the resulting construct
Transfection into cells was performed.

293T cells had 10% FCS.
D containing 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin
Cultured in MEM medium.

Transient transfection of the above-described construct was carried out by the lipofection method using Lipofectamine 2000 (manufactured by Gibco BRL). The number of transfected cells obtained was 10
After culturing in% FCS / DMEM overnight, the obtained cells were subjected to fluorescence microscope analysis.

As a result, as shown in FIG. 3, the ectopically expressed EGFP-PKACβ4ab was localized mainly in the cytoplasm but not in the nucleus, while GFP-PKACβ was absent.
1 was found to be diffusely distributed in the cells. These results suggest that PKACβ4ab may have a special function in the brain.

Example 3 From the interaction between p75 ^NTR and the catalytic subunit of PKA, it was verified that p75 ^NTR was a substrate for PKA.

The p75ICD coding sequence was transformed into pGEX-5X.
A recombinant vector for expressing a glutathione-S-transferase (GST) fusion protein was constructed by inserting it into the cloning site of a bacterial expression vector [manufactured by Amersham Pharmacia Biotech].

Using the recombinant vector, E. coli DH5
α was transformed. The transformant obtained had an OD ₆₀₀ of
After growing the cells to 0.6, 1 mM isopropyl-1-thio-β- was added to induce protein synthesis.
D-galactopyranoside (IPTG) was added to the culture and the cells were grown at 25 ° C for a further 16 hours. A cell extract was prepared from the obtained culture, and the cell extract was subjected to glutathione affinity chromatography to obtain p75.
The ICD GST fusion protein was purified. The purified protein was incubated with Factor Xa overnight at 4 ° C., then the resulting product was subjected to ion exchange HPLC on a DEAE cellulose column to remove the GST moiety, thereby yielding p75ICD.

The obtained p75ICD (40 μg) was added to 2
0 mM HEPES (pH 7.5) and 10 mM MgCl ₂
And 5 mM dithiothreitol and 100 mM Na
Cl and 1 mM [γ- ³² P] ATP (0.5 Ci / m
50 μl by adding to a PKA reaction buffer containing
And the total volume of the solution was 50 U and incubated with 50 U of PKA catalytic subunit [Calbiochem] at 30 ° C. for 30 minutes.

To verify the specificity, 1 μg of PKA inhibitor (PKI) [Sigma] was added to the reaction buffer to specifically inhibit the activity of PKA. 100 ng histone protein [Sigma (Sig
ma)] was used as a positive control.

As a result, as shown in FIG. 4, wild-type p7
For 5 ICD incubation, 22 kDa (p7
It can be seen that ³² P-labeled molecular species appeared in (the expected molecular weight of 5ICD) and its radioactivity was completely reduced by PKI which is a specific inhibitor of PKA. This is p75IC
It is shown that D is phosphorylated by PKA in vitro.

The amino acid sequence R / KNNS / T is the consensus PKA recognition site, and S or T is the phosphorylation site. The human p75ICD sequence has one putative PKA phosphorylation site containing serine 304 in the juxtamembrane linker region. As shown in FIG. 5, the PKA recognition site is also conserved in mouse p75ICD and chicken p75ICD.

To determine if this conserved site is phosphorylated by PKA, we introduced a serine to glycine substitution mutation at this site in the p75ICD sequence to generate the p75ICD mutant (p75ICDm) protein. did.

Specifically, a substitution mutation from serine to glycine at the PKA phosphorylation consensus site of p75ICD was introduced by PCR using a mutation-introducing primer. p75EXDm coding sequence to pGEX-5X
Cloning into a bacterial expression vector [Amersham Pharmacia Biotech], glutathione-S-transferase (GST)
A fusion protein was made. Then, the p75ICD
By the same operation as above, p75ICDm was obtained.

The obtained p75ICDm (40 μg) was added to
20 mM HEPES (pH 7.5) and 10 mM MgCl
₂ and 5 mM dithiothreitol and 100 mM N
aCl and 1 mM [γ- ³² P] ATP (0.5 Ci /
50 μm by adding to a PKA reaction buffer containing
The total liquid volume was 1 and the cells were incubated with 50 U of PKA catalytic subunit [Calbiochem] at 30 ° C. for 30 minutes.

In order to verify the specificity, 1 μg of PKA inhibitor (PKI) [Sigma (Sigma)] was added to the reaction buffer to specifically inhibit PKA activity. 100 ng histone protein [Sigma (Sig
ma)] was used as a positive control.

As a result, as shown in FIG. 4, p75IC
It can be seen that Dm is not phosphorylated by PKA. Therefore, it was revealed that the conserved site is the only site in p75ICD that is phosphorylated by PKA.

Example 4 Phosphorylation of p75 ^NTR is expected to play a role in the efficiency of signal transduction or its membrane localization. p75
^NTR is to regulate a wide range of signal was verified the possibility to utilize the compartments to set a specific downstream signaling molecules. Specifically, activated p75 ^NTR
Were clustered within lipid rafts, a structure of signaling organelles.

[0100] To evaluate the effect of p75 ^{NTR on} membrane localization when NGF was used without considering TrkA activation, mouse cerebellar neurons that express p75 ^NTR but not TrkA were used.

Cerebellum was removed from P6 mouse, and 0.25%
It was put in trypsin and ground. The product obtained is 20
Incubated with 0 U / ml DNase I for 15 minutes at 37 ° C. DMEM containing 10% FCS was added to the obtained product, and the mixture was subjected to centrifugation at 1,000 rpm for 5 minutes. The obtained cells were treated with 1 mg / ml BSA.
And 100 μg / ml transferrin, 30 nM selenium, 10 μg / ml insulin, and 0.1 nM L-
Resuspending in Sato medium containing thyroxine gave a single cell suspension.

10 cm tissue culture dish, 4 chamber slide and 6 well culture dish at 10 μg / m
It was coated with 1 l of poly-L-lysine [Sigma] for 1 hour at room temperature. Excess poly -L- lysine washed out with H ₂ O, and washed dishes again.

The isolated neurons were seeded on the poly-L-lysine coated dishes at a density of 5 × 10 ⁷ cells / dish. 24 hours after dissociation and every 48 hours,
The medium was replaced with Sato medium.

A part of the obtained cerebellar granule neurons was treated with 200 nM of protein kinase A inhibitor KT57.
Pretreatment with 20 [Calbiochem] was carried out for 1 hour, and the rest was not subjected to the pretreatment.

NGF was added 20 minutes before lysis of the cells.
Added at a concentration of 0 ng / ml. Next, 0.5% Brij
-58, 100 μg / ml orthovanadate and an extraction buffer solution [composition: 10 mM Tris-HCl (pH 7.5)] containing a protease inhibitor cocktail [Roche].
150mM NaCl, 5mM EDTA] 0.5m
Cells were lysed on ice with 1.

To the extract obtained, 0.5 ml of 80%
Sucrose was added to adjust to 40% sucrose. Then, the obtained mixture was transferred to a SW41Ti ultracentrifugation tube (manufactured by Beckman), and 8 ml of the extraction buffer containing 30% sucrose and 1 ml of distilled water were further overlaid. All the operations were performed on ice in a low temperature room at 4 ° C. Centrifuge (16 hours, 20
20,000 × g, 4 ° C.), 0.83 ml of each 12 fractions were collected from the upper end of the ultracentrifugation tube, and an equal amount thereof was prepared for SDS-PAGE and immunoblot analysis. . p75 ^NTR and Flotillin
in) 1 was detected using a polyclonal anti-human p75 antibody [1: 2000, manufactured by Promega] and a monoclonal anti-rat flotillin 1 antibody [1: 1000, manufactured by Transduction Lab.].
Used respectively.

As described above, Brij-58 insoluble low-density membranes (raft-binding proteins) were separated from fully soluble high-density membranes (non-raft-binding proteins) and cytoskeletal proteins using buoyancy gradient and p75 by SDS-PAGE. ^NTR immunoreactivity was analyzed. The result is shown in FIG.

As a result, stimulation with NGF (200 ng / ml) resulted in a significant amount of p75 ^NTR to the top of the buoyancy gradient.
, Which confirmed its connection with low-density lipids. TrkB was present only in the soluble membrane fraction with or without NGF addition. These data indicate that NGF induces p75 ^NTR translocation into lipid rafts.

Example 5 The distribution of p75 ^NTR with respect to lipid rafts was examined by staining E18 rat hippocampal neurons with rhodamine labeled cholera toxin B subunit (CT-B). CT-
B is ganglioside G among glycosphingolipids.
It has a strong affinity for M1 and a weak affinity for other gangliosides. Therefore, although non-raft GM1 is also detected, it can be used as a marker for lipid rafts rich in glycosphingolipids. Specifically, the distribution of p75 ^NTR was examined by the following ^procedure .

The hippocampus was excised from E19 rat and 40 U /
With ml papain and 200U / ml DNase I,
Incubated at 37 ° C for 15 minutes. Then, DMEM containing 10% FCS was added to the obtained product.
The resulting mixture was subjected to centrifugation at 1,000 rpm for 5 minutes. The obtained cells were treated with DM containing 10% FCS.
It was resuspended in EM to obtain hippocampal neurons.

The isolated neuron was transformed into the poly-L-
Lysine-coated dish, 1 x 10 for immunofluorescence analysis
Seed at a density of ⁵ cells / chamber.

24 hours after isolation and every 48 hours, the medium was replaced with DMEM containing B27 supplement. After isolation, it was incubated for at least 3 days.

Labeling of endogenous glycosphingolipids was performed as described previously, using CT-B-rhodamine conjugate [List Biological Laboratories (List.
Biological Laboratories)] in phosphate buffered saline (PBS) containing 0.1% BSA.
Used at a concentration of / ml.

After CT-B labeling, cells were mixed with anti-CT-B antibody (PBS containing 0.1% BSA; Calbiochem).
(Calbiochem)] and incubated at 37 ° C. for 20 minutes to induce aggregation or patch formation of lipid rafts. Neurons were treated with 200 ng / ml NGF for 10 minutes and fixed.

To perform immunofluorescent staining, the cells were 4%
The cells were fixed with paraformaldehyde-containing PBS at room temperature for 30 minutes. The cells were then blocked with PBS containing 1% BSA and 5% goat serum for 1 hour. next,
The cells were incubated overnight at 4 ° C. with MC-192 anti-p75 ^NTR extracellular domain monoclonal antibody (40 μg / ml in PBS containing 1% BSA and 5% goat serum; donated by Dr. H. Hatanaka). The cells were washed with PBS and then incubated with a fluorescein-conjugated secondary antibody (Molecular Probes) for 1 hour at room temperature. The obtained product is
It was observed with a confocal fluorescence microscope. As a confocal fluorescence microscope, a confocal microscope LSM510 manufactured by Zeiss Co. equipped with a 40 × objective lens was used.

As a result, as shown in FIG. 7, the staining was concentrated in the characteristic patch in the membrane. Endogenous p75 ^NTR and C
Co-localization with TB was confirmed by double immunofluorescence in hippocampal neurons.

Next, it was examined whether or not cAMP-PKA is an essential component when p75 ^NTR transfers to lipid rafts.

Prior to the buoyancy gradient analysis, the PKA inhibitor KT5720 (200 nM) was added to the culture medium of cerebellar neurons. As a result, as shown in FIG.
In the presence of KT5720, stimulation with NGF did not increase the amount of p75 ^NTR translocated to lipid rafts.
These data indicate that PKA activation is essential for NGF-induced translocation of p75 ^NTR into lipid rafts.

Example 6 Elevated intracellular cAMP levels trigger signaling pathways through activation of PKA. Therefore, p75 ^NTR
To investigate whether itself could induce activation of cAMP-PKA, cA in primary cerebellar neurons was investigated.
The intracellular level of MP was measured.

The neurons were seeded on the poly-L-lysine-coated dish at a density of 5 × 10 ⁷ cells / dish for the raft fraction isolation for the cAMP assay. Seed cerebellar neurons in a 6-well dish,
Incubated for at least 3 days. 200 NGF
Cells were lysed after addition at a concentration of ng / ml and incubation for 0-30 minutes. Intracellular cyclic AMP
(CAMP) was determined by a competitive immunoassay by the manufacturer [Amersham Pharmacia Biotech (Amers
ham Pharmacia Biotech)].
In addition, cAMP measured by competitive immunoassay
The basal level of the was in the range of 20-30 fmol / 10 ⁶ cells.

As a result, as shown in FIG. 9, 200 ng
Intracellular cAMP when NGF was added at a concentration of 1 / ml
The amount increased about 1.6 times after 10 minutes, and after 30 minutes,
It turned out that it returned to almost the basal level. Therefore, p
It was found that ligand binding to the 75 ^NTR can activate PKA.

NGF was added to RhoA in cells expressing endogenous p75 ^NTR and transfected p75 ^NTR.
This GTP is one of the biological consequences of activation of p75 ^NTR as it causes a loss of activation.
I chose Aase. Rho activity was measured using the Rho binding domain of the effector protein Rhotekin. Since Rotekin, which is a downstream target of Rho, specifically binds to GTP-bound Rho, the use of this Rotekin allows the active Rho to be precipitated and quantified.

Cerebellar neurons were grown in 10 cm tissue culture plates. 1 by 200 nM KT5720
NGF was added at a concentration of 200 ng / ml for 10 minutes with or without time pretreatment.

Next, 1% Triton X-100 and 0.
5% sodium deoxycholate and 0.1% SD
S, 500 mM NaCl, 10 mM MgCl ₂
The cells were lysed in 50 mM Tris, pH 7.5, containing a protease inhibitor cocktail. The obtained cell lysate was subjected to centrifugation at 13,000 × g, 4 ° C. for 10 minutes. The resulting supernatant was incubated with 20 μg of GST-Rotekin Rho binding domain beads for 45 minutes at 4 ° C. Then, the beads were washed with a washing buffer [1% Triton X-100, 150 mM NaCl, 10 mM M].
It was washed 4 times with gCl ₂ , protease inhibitor cocktail, 50 mM Tris (pH 7.5)]. The bound Rho protein is a monoclonal antibody (Santa Santa) against RhoA.
Cruz Biotechnology) was used for detection by Western blotting.

As a result, the level of active RhoA was found to be high after the addition of NGF (50 ng / ml) to cerebellar neurons.
It decreased within 10 minutes, which was consistent with the previous report. In addition, FIG.
As shown in, KT57 was added 1 hour before the addition of NGF.
When cells were incubated with 20 (200 nM), KT5720 itself had no effect on Rho activity but completely blocked this inactivation. These results indicate that PKA activation is induced by ligand binding to p75 ^NTR and that PKA activation is essential for p75 ^NTR- mediated RhoA inactivation.

Sequence Listing Free Text SEQ ID NO: 3 is a primer sequence for amplifying a PKACβ4ab-derived fragment.

SEQ ID NO: 4 is a primer sequence for amplifying a PKACβ4ab-derived fragment.

SEQ ID NO: 5 is the sequence of p75ICDm.

[0129]

INDUSTRIAL APPLICABILITY According to the modulator of the present invention, it is possible to regulate the signal transduction mediated by the neurotrophin receptor p75, and thereby the neurotrophin receptor p7.
It has an excellent effect of being able to treat or prevent a disease caused by an abnormality in signal transduction mediated by 5. Furthermore, the therapeutic or prophylactic pharmaceutical composition of the present invention is useful for the treatment or prevention of diseases such as neurodegenerative diseases, central nerve damage, neurogenic tumors and the like.

[0130]

[Sequence list]                                SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> An agent for controlling neurotrophin receptor-mediated signal tra nsduction <130> KJ-14-001 <160> 8 <170> PatentIn Ver. 2.1 <210> 1 <211> 4595 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (327) .. (1388) <400> 1 acattttctc cctgccattt tgagttgttc tagtggtata tgaaggaggc tgggataact 60 agcttgaaag aaattcagtc tagttataga catctttggc attaatctga tgtttactag 120 tgatatctca tgctaggcag ttatgctttg cttctagggg cttctctttt taaaacaaaa 180 gaaagctctt ttcgttttct gtgtgctgca tgctccagtg tgtgtgttta caccatcggt 240 tcttctccct ctagagatta gcataactcc ctttgctgtt ggattgttat tttgagcaat 300 atgttttgga aaggttggtt ttcatc atg agt gca cgc aaa tca tca gat gca 353                              Met Ser Ala Arg Lys Ser Ser Asp Ala                                1 5 tct gct tgc tcc tct tca gaa ata tct gtg aaa gag ttt cta gcc aaa 401 Ser Ala Cys Ser Ser Ser Glu Ile Ser Val Lys Glu Phe Leu Ala Lys  10 15 20 25 gcc aaa gaa gac ttt ttg aaa aaa tgg gag aat cca act cag aat aat 449 Ala Lys Glu Asp Phe Leu Lys Lys Trp Glu Asn Pro Thr Gln Asn Asn                  30 35 40 gcc gga ctt gaa gat ttt gaa agg aaa aaa acc ctt gga aca ggt tca 497 Ala Gly Leu Glu Asp Phe Glu Arg Lys Lys Thr Leu Gly Thr Gly Ser              45 50 55 ttt gga aga gtc atg ttg gta aaa cac aaa gcc act gaa cag tat tat 545 Phe Gly Arg Val Met Leu Val Lys His Lys Ala Thr Glu Gln Tyr Tyr          60 65 70 gcc atg aag atc tta gat aag cag aag gtt gtt aaa ctg aag caa ata 593 Ala Met Lys Ile Leu Asp Lys Gln Lys Val Val Lys Leu Lys Gln Ile      75 80 85 gag cat act ttg aat gag aaa aga ata tta cag gca gtg aat ttt cct 641 Glu His Thr Leu Asn Glu Lys Arg Ile Leu Gln Ala Val Asn Phe Pro  90 95 100 105 ttc ctt gtt cga ctg gag tat gct ttt aag gat aat tct aat tta tac 689 Phe Leu Val Arg Leu Glu Tyr Ala Phe Lys Asp Asn Ser Asn Leu Tyr                 110 115 120 atg gct atg gaa tat gtc cct ggg ggt gaa atg ttt tca cat cta aga 737 Met Ala Met Glu Tyr Val Pro Gly Gly Glu Met Phe Ser His Leu Arg             125 130 135 aga att gga agg ttc agt gag ccc cat gca cgg ttc tat gca gct can 785 Arg Ile Gly Arg Phe Ser Glu Pro His Ala Arg Phe Tyr Ala Ala Thr         140 145 150 ata gtg cta aca ttc gag tac ctc cat tca cta gac ctc atc tac aga 833 Ile Val Leu Thr Phe Glu Tyr Leu His Ser Leu Asp Leu Ile Tyr Arg     155 160 165 gat cta aaa cct gaa aat ctc tta att gac cat caa ggc tat atc cag 881 Asp Leu Lys Pro Glu Asn Leu Leu Ile Asp His Gln Gly Tyr Ile Gln 170 175 180 185 gtc aca gac ttt ggg ttt gcc aaa aga gtt aaa ggc aga act tgg aca 929 Val Thr Asp Phe Gly Phe Ala Lys Arg Val Lys Gly Arg Thr Trp Thr                 190 195 200 tta tgt gga act cca gag tat ttg gct cca gaa ata att ctc agc aag 977 Leu Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Ile Ile Leu Ser Lys             205 210 215 ggc tac aat aag gca gtg gat tgg tgg gca tta gga gtg cta atc tat 1025 Gly Tyr Asn Lys Ala Val Asp Trp Trp Ala Leu Gly Val Leu Ile Tyr         220 225 230 gaa atg gca gct ggc tat ccc cca ttc ttt gca gac caa cca att cag 1073 Glu Met Ala Ala Gly Tyr Pro Pro Phe Phe Ala Asp Gln Pro Ile Gln     235 240 245 att tat gaa aag att gtt tct gga aag gtc cga ttc cca tcc cac ttc 1121 Ile Tyr Glu Lys Ile Val Ser Gly Lys Val Arg Phe Pro Ser His Phe 250 255 260 265 agt tca gat ctc aag gac ctt cta cgg aac ctg ctg cag gtg gat ttg 1169 Ser Ser Asp Leu Lys Asp Leu Leu Arg Asn Leu Leu Gln Val Asp Leu                 270 275 280 acc aag aga ttt gga aat cta aag aat ggt gtc agt gat ata aaa act 1217 Thr Lys Arg Phe Gly Asn Leu Lys Asn Gly Val Ser Asp Ile Lys Thr             285 290 295 cac aag tgg ttt gcc acg aca gat tgg att gct att tac cag agg aag 1265 His Lys Trp Phe Ala Thr Thr Asp Trp Ile Ala Ile Tyr Gln Arg Lys         300 305 310 gtt gaa gct cca ttc ata cca aag ttt aga ggc tct gga gat acc agc 1313 Val Glu Ala Pro Phe Ile Pro Lys Phe Arg Gly Ser Gly Asp Thr Ser     315 320 325 aac ttt gat gac tat gaa gaa gaa gat atc cgt gtc tct ata aca gaa 1361 Asn Phe Asp Asp Tyr Glu Glu Glu Asp Ile Arg Val Ser Ile Thr Glu 330 335 340 345 aaa tgt gca aaa gaa ttt ggt gaa ttt taaagaggaa caagatgaca 1408 Lys Cys Ala Lys Glu Phe Gly Glu Phe                 350 tctgagctca cactcagtgt ttgcactctg ttgagagata aggtagagct gagaccgtcc 1468 ttgttgaagc agttacctag ttccttcatt ccaacgactg agtgaggtct ttattgccat 1528 catccgtgtg cgcactctgc atccacctat gtaacaaggc accgctaagc aagcattgtc 1588 tgtgccataa cacagtacta gaccactttc ttacttctct ttgggttgtc tttctcctct 1648 cctatatcca tttcttcctt ttcaatttca ttggttttct ctaaacagtg ctccatttta 1708 ttttgttggt gtttcagatg ggcagtgtta tggctacgtg atatttgaag ggaaggataa 1768 gtgttgcttt cagtagttat tgccaatatt gttgttggtc aatggcttga agataaactt 1828 tctaataatt attatttctt tgagtagctc agacttggtt ttgccaaaac tcttggtaat 1888 ttttgaagat agactgtctt atcaccaagg aaatttatac aaattaagac taactttctt 1948 ggaattcact attctggcaa taaattttgg tagactaata cagtacagct agacccagaa 2008 atttggaagg ctgtagatca gaggttctag ttccctttcc ctccttttat atcctcctct 2068 ccttgagtaa tgaagtgacc agcctgtgta gtgtgacaaa cgtgtctcat tcagcaggaa 2128 aaactaatga tatggatcat cacccagatt ctctcacttg gtaccagcat ttctgtaggt 2188 attagagaag agttctaagt tttctaaacc ttaactgttc cttaaggatt ttagccagta 2248 ttttaataga acatgattaa tgaaagtgac aaattttaaa ttttctctaa tagtcctcat 2308 cataaacttt ttaaaggaaa ataagcaaac taaaaagaac attggtttag ataaatactt 2368 atactttgca aagtcaaaaa tggcttgatt tttggaaaca atatagaggt attcatattt 2428 aaatgagggt ttacatttgt tttgttttgt aaccgttaaa aagaagttgt ttccagctaa 2488 ttattgtggt gtactatatt tgtgagccta gggtaggggc actgctgcaa cttctgcttt 2548 catcccatgc ctcatcaatg aggaaaggga acaaagtgta taaaacctgc cacaattgta 2608 ttttaatttt gaggtatgat attttcagat atttcataat ttctaacctc tgttctctca 2668 gtaaacagaa tgtctgatcg atcatgcaga tacaatgttg gtatttgaga ggttagtttt 2728 tttcctacac ttttttttgc caactgactt aacaacattg ctgtcaggtg gaaatttcaa 2788 gcacttttgc acatttagtt cagtgtttgt tgagaatcca tggcttaacc cacttgtttt 2848 gctatttttt tctttgcttt taattttccc catctgattt tatctctgcg tttcagtgac 2908 ctaccttaaa acaacacacg agaagagtta aactgggttc attttaatga tcaatttacc 2968 tgcatataaa atttattttt aatcaagctg atcttaatgt atataatcat tctatttgct 3028 ttattatcgg tgcaggtagg tcattaacac cacttctttt catctgtacc acaccctggt 3088 gaaacctttg aagacataaa aaaaacctgt ctgagatgtt ctttctacca atctatatgt 3148 ctttcggtta tcaagtgttt ctgcatggta atgtcatgta aatgctgata ttgatttcac 3208 tggtccatct atatttaaaa cgtgcaagaa aaaaataaaa tactctgctc tagcaagttt 3268 tgtgtaacaa aggcatatcg tcatgttaat aaatttaaaa catcattcgt ataaaatatt 3328 ttaattttct tgtatttcat ttagacccaa gaacatgctg accaatgtgt tctatatgta 3388 aactacaaat tctatggtag ctttgttgta tattattgta aaattatttt aataagtcat 3448 ggggatgaca atttgattat tacaatttag ttttcagtaa tcaaaaagat ttctatgaat 3508 tctaaaaaat atttttttct atgaaattac tagtgcccag ctgtagaatc taccttaggt 3568 agatgatccc tagacatacg ttggttttga gggctattca gccattccat tttactctct 3628 atttaaaggc cgtgagcaag cttgtcatga gcaaatatgt caagggagtc aatttctgac 3688 caatcaagta cactaaatta gaatattttt aaagtatgta acattcccag tttcagccac 3748 aatttagcca agaataagat aaaaacttga ataagaagta agtagcataa atcagtattt 3808 aacctaaaat tacatatttg aaacagaaga tattatgtta tgctcagtaa ataattaaga 3868 gatggcattg tgtaagaagg agccctagac tgaaagtcaa gacatctgaa tttcaggctg 3928 gaaaactatc agtatgatct cagcctcagt tctcttgtct gtaaaatgga agaactggat 3988 taggcagttt gtaagattcc tcctaacttt cacagtcgat gacaagattg tctttttatc 4048 tgatattttg aagggtatat tgctttgaag taagtctcaa taaggcaata tattttaggg 4108 catctttctt cttatctctg acagtgttct taaaattatt tgaatatcat aagagccttg 4168 gtgtctgtcc taattccttt ctcactcacc gatgctgaat acccagttga atcaaactgt 4228 caacctacca aaaacgatat tgtggcttat gggtattgct gtctcattct tggtatattc 4288 ttgtgttaac tgcccattgg cctgaaaata ctcattgtaa gcctgaaaaa aaaaatcttt 4348 cccactgttt tttctgcttg ttgtaagaat caaatgaaat aatgtatgtg aaagcacctt 4408 gtaaactgta acctatcaat gtaaaatgtt aaggtgtgtt gttatttcat taattacttc 4468 tttgtttaga atggaatttc ctatgcacta ctgtagctag gaaatgctga aaacaactgt 4528 gttttttaat taatcaataa ctgcaaaatt aaagtacctt caatggaaaa aaaaaaaaaa 4588 aaaaaaa 4595

[0131] <210> 2 <211> 354 <212> PRT <213> Homo sapiens <400> 2 Met Ser Ala Arg Lys Ser Ser Asp Ala Ser Ala Cys Ser Ser Ser Glu   1 5 10 15 Ile Ser Val Lys Glu Phe Leu Ala Lys Ala Lys Glu Asp Phe Leu Lys              20 25 30 Lys Trp Glu Asn Pro Thr Gln Asn Asn Ala Gly Leu Glu Asp Phe Glu          35 40 45 Arg Lys Lys Thr Leu Gly Thr Gly Ser Phe Gly Arg Val Met Leu Val      50 55 60 Lys His Lys Ala Thr Glu Gln Tyr Tyr Ala Met Lys Ile Leu Asp Lys  65 70 75 80 Gln Lys Val Val Lys Leu Lys Gln Ile Glu His Thr Leu Asn Glu Lys                  85 90 95 Arg Ile Leu Gln Ala Val Asn Phe Pro Phe Leu Val Arg Leu Glu Tyr             100 105 110 Ala Phe Lys Asp Asn Ser Asn Leu Tyr Met Ala Met Glu Tyr Val Pro         115 120 125 Gly Gly Glu Met Phe Ser His Leu Arg Arg Ile Gly Arg Phe Ser Glu     130 135 140 Pro His Ala Arg Phe Tyr Ala Ala Thr Ile Val Leu Thr Phe Glu Tyr 145 150 155 160 Leu His Ser Leu Asp Leu Ile Tyr Arg Asp Leu Lys Pro Glu Asn Leu                 165 170 175 Leu Ile Asp His Gln Gly Tyr Ile Gln Val Thr Asp Phe Gly Phe Ala             180 185 190 Lys Arg Val Lys Gly Arg Thr Trp Thr Leu Cys Gly Thr Pro Glu Tyr         195 200 205 Leu Ala Pro Glu Ile Ile Leu Ser Lys Gly Tyr Asn Lys Ala Val Asp     210 215 220 Trp Trp Ala Leu Gly Val Leu Ile Tyr Glu Met Ala Ala Gly Tyr Pro 225 230 235 240 Pro Phe Phe Ala Asp Gln Pro Ile Gln Ile Tyr Glu Lys Ile Val Ser                 245 250 255 Gly Lys Val Arg Phe Pro Ser His Phe Ser Ser Asp Leu Lys Asp Leu             260 265 270 Leu Arg Asn Leu Leu Gln Val Asp Leu Thr Lys Arg Phe Gly Asn Leu         275 280 285 Lys Asn Gly Val Ser Asp Ile Lys Thr His Lys Trp Phe Ala Thr Thr     290 295 300 Asp Trp Ile Ala Ile Tyr Gln Arg Lys Val Glu Ala Pro Phe Ile Pro 305 310 315 320 Lys Phe Arg Gly Ser Gly Asp Thr Ser Asn Phe Asp Asp Tyr Glu Glu                 325 330 335 Glu Asp Ile Arg Val Ser Ile Thr Glu Lys Cys Ala Lys Glu Phe Gly             340 345 350 Glu Phe

[0132] <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: a squence of primer for amplif ying a fragment derived from PKACbeta4ab. <400> 3 ccctgccatt ttgagttgtt c 21

[0133] <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: a squence of primer for amplif ying a fragment derived from PKACbeta4ab. <400> 4 tatttctgaa gaggagcaag c 21

[0134] <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: a sequence of p75ICDm <400> 5 Lys Leu His Gly   1

[0135] <210> 6 <211> 8 <212> PRT <213> Mouse <400> 6 Gly Glu Lys Leu His Ser Asp Ser   1 5

[0136] <210> 7 <211> 8 <212> PRT <213> Homo sapiens <400> 7 Gly Glu Lys Leu His Ser Asp Ser   1 5

[0137] <210> 8 <211> 8 <212> PRT <213> Allus gallus domesticus <400> 8 Gly Glu Lys Leu His Ser Asp Ser   1 5

[Brief description of drawings]

FIG. 1 is a diagram showing the interaction between p75 ^NTR and PKACβ in the yeast two-hybrid system.
The complementarity of the cdc25-H temperature sensitive mutation was tested. Co-transforming the indicated plasmids into yeast, isolating 3 independent colonies for each plasmid combination,
It was grown at 25 ° C, a replica plate was prepared on a galactose plate, and grown at 37 ° C. Yeast transformed with Ras family proteins grew independently of protein-protein interactions.

FIG. 2 is a diagram showing tissue distribution of PKACβ4ab mRNA. Human multiple tissue Northern blot (human multiple tissure Northern (MTN) bl
ot; manufactured by Clontech)]
A ³² P-labeled 370 bp fragment corresponding to exon 1-4, exon 1-a and exon 1-b of Cβ4ab was used as a probe for analysis.

FIG. 3 is a diagram showing intracellular localization of PKACβ variants. 293T cells (1 × 10 ⁴ cells / well) grown on a 4-well chamber slide were transferred to PKA.
Cβ1-GFP (left panel) or PKACβ4ab-G
Transfected by FP (right panel). 36 hours after transfection, the cells were observed under a confocal microscope (40 ×).

FIG. 4 is a diagram showing in vitro phosphorylation of p75 ^NTR . Wild-type p75 ^NTR intracellular domain (p75ICD) or mutant p75 ^NTR intracellular domain (p75ICDm) expressed by bacteria was [γ- ³² P] AT
Incubation was carried out for 30 minutes in the presence or absence of the catalytic subunit of PKA and PKI in the presence of P. The resulting reaction mixture was separated by SDS-PAGE and the gel was exposed to X-ray film or imaging plate.

FIG. 5 shows the amino acid sequence of the PKA phosphorylation site of p75ICD. p75 ^{NT R} has one of PKA phosphorylation consensus site at juxtamembrane site linker region (KLHS) sequence surrounding the site, murine (SEQ ID NO: 6), chicken (SEQ ID NO: 8) and human ( It is highly conserved in SEQ ID NO: 7). p7
The 5ICD mutant (p75ICDm) has the serine at position 304 replaced with glycine at the consensus site.

FIG. 6 is a diagram showing the kinetics of p75 ^NTR when stimulated with NGF. Mouse cerebellar neurons (P6)
10 in the presence or absence of GF (200 ng / ml)
Processed for a minute. The neurons were then detergent extracted and the resulting lysates were subjected to sucrose density gradient centrifugation. Gradient fractions from top to bottom (# 1- # 1
2) Recovered. Equal amount of insoluble raft fraction (# 2 and #
3) and soluble high density fractions (# 11 and # 12) in SDS
-Subjected to PAGE. Western blots of p75 ^NTR , TrkB and Flotillin-1 are shown. In unstimulated cells, p75 ^NTR fractionates mainly in the soluble fraction. After NGF stimulation, many parts of the p75 ^NTR , particularly the molecular weight band in the middle, float in the raft fraction in which the raft marker Flotillin-1 is fractionated. TrkB did not translocate in parallel with p75 ^NTR .

FIG. 7 shows CT-B and p in hippocampal neurons.
It is a figure which shows the colocalization with 75 ^NTR . The neuron is C
Aggregated lipid rafts were visualized with TB-rhodamine and anti-CT-B, then fixed and stained with anti-p75 ^NTR antibody and Alexa fluor ^™ 488 conjugated secondary antibody. Optical sectional view of p75 ^NTR alone [panel a,
d; indicated by "p75 ^NTR " in the figure], an optical cross-sectional view of CT-B alone [b, e; indicated by "CTB (Raft)" in the figure] and images at the p75 ^NTR and CT-B. The superimposed images [panels c and f; indicated by "Merge" in the figure] are shown. The bottom panel is a high magnification image of the top panel. The p75 ^NTR is localized in aggregated lipid rafts, especially in the axon branch horns (arrowheads).

FIG. 8 shows PKA-dependent translocation of p75 ^NTR into lipid rafts. P6 mouse cerebellar neurons untreated (control), treated with NGF for 10 minutes [“NGF (10 min)” in the figure], 200 nM P
What was treated with KA inhibitor KT5720 for 1 hour [in the figure,
"KTF5720 (200 nM)"], P of 200 nM
Pre-treatment with KA inhibitor KT5720 for 1 hour and NGF
What was processed for 10 minutes was prepared. For each, the insoluble raft fraction and the soluble high density fraction were isolated,
It was analyzed by Western blot.

FIG. 9 shows intracellular cAM in cerebellar neurons.
It is a figure which shows the result of the time measurement of P. 5 × 10 ⁶ neurons were separated into each well of a 6-well dish. Treatments were with NGF (200 nM) for 0-30 minutes as indicated, then cAMP levels were measured and compared to standards. Results are the average of 3 experiments.

FIG. 10 is a diagram showing affinity precipitation of RhoA in P6 mouse cerebellar neurons. NGF is R
Decreases the activity of hoA. Inactivation is 200 nM K
Blocked in neurons pretreated with T5720 for 1 hour.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 25/00 A61P 25/28 4H045 25/28 35/00 35/00 43/00 111 43/00 111 C07K 16/40 // C07K 16/40 C12N 9/12 C12N 9/12 A61K 37/02 15/09 ZNA C12N 15/00 ZNAA F term (reference) 4B024 AA01 BA10 CA04 DA02 DA05 DA11 EA04 FA02 GA11 HA01 HA03 4B050 CC03 CC07 DD11 LL01 4C084 AA02 AA07 AA13 BA01 BA08 BA22 CA53 DC50 MA24 MA66 NA10 ZA012 ZA162 ZB262 4C085 AA13 AA14 BB11 BB41 CC23 DD61 DD88 EE01 GG02 GG03 GG04 4C086 AA01 AA02 A20 A20A20 A20A20 A20A20 A20A20 A20A16 A16 A16A16 A16A16 A16A16 A16A16A16A20

Claims (7)

[Claims] 1. An amino acid sequence represented by the following (a) to (f): (a) the amino acid sequence shown in SEQ ID NO: 2, (b) the amino acid sequence having at least 97% sequence identity with said SEQ ID NO: 2. (C) substitution of at least one amino acid residue,
An amino acid sequence different from said SEQ ID NO: 2 through deletion, addition or insertion, (d) in (c) above, amino acid numbers: 4, 5, 6, 7, 10, 10 in said SEQ ID NO: 2
Amino acid sequence in which 2, 13, 14 and 15 are conserved, (e) the sequence of any one of (a) to (d) through at least one amino acid residue that imparts physiologically equivalent physicochemical properties An amino acid sequence different from the above, and (f) in the living body or in a physicochemical environment equivalent to the living body,
To an amino acid sequence capable of supplying a polypeptide having a physiologically equivalent three-dimensional structure to the polypeptide consisting of the amino acid sequence of (e), and a neurotro Fin receptor p75
(Gene bank accession number: NM — 002507), which is a regulator of signal transduction mediated by neurotrophin receptor p75, containing a polypeptide having an activity of phosphorylating (Gene Bank Accession No. NM — 002507). 2. A regulator of signal transduction mediated by the neurotrophin receptor p75, which comprises as an active ingredient an antibody or a fragment thereof that specifically binds to the polypeptide of claim 1. 3. A regulator of signal transduction mediated by the neurotrophin receptor p75, which comprises as an active ingredient a nucleic acid encoding the polypeptide of claim 1. 4. The nucleic acid comprises the following (A) to (F): (A) a base sequence represented by SEQ ID NO: 1, (B) a base sequence different from the SEQ ID NO: 1 by degeneracy, ( C) a nucleotide sequence having a sequence identity of at least 97% with said SEQ ID NO: 1, and the encoded polypeptide exhibits p75 phosphorylation ability, (D) the base in said SEQ ID NO: 1 Nos .: 336 to 347, base numbers: 354 to 356, and base numbers: 360 to 371 are conserved, and differ from SEQ ID NO: 1 through substitution, deletion, addition or insertion of at least one base, and The encoded polypeptide is a nucleotide sequence that exhibits phosphorylation ability of p75, (E) is a nucleotide sequence of a nucleic acid that can hybridize to a complementary strand of the nucleic acid consisting of SEQ ID NO: 1 under stringent conditions. And the encoded polypeptide is p7 A nucleotide sequence that exhibits phosphorylation ability of 5 and (F) a nucleic acid polymorphism that differs from SEQ ID NO: 1 and is encoded by a polypeptide that exhibits p75 phosphorylation ability. The regulator according to claim 3, which is a nucleic acid containing a base sequence selected from the group consisting of a certain base sequence. 5. A regulator of signal transduction mediated by the neurotrophin receptor p75, which comprises, as an active ingredient, an antisense nucleic acid having a sequence complementary to the nucleic acid described in claim 3 or 4. 6. A medicament for treating or preventing a disease caused by an abnormality in signal transduction mediated by neurotrophin receptor p75, which comprises the modulator according to any one of claims 1 to 5 as an active ingredient. Composition. 7. The treatment or prevention according to claim 6, wherein the disease caused by abnormal signal transduction through the neurotrophin receptor p75 is a disease selected from the group consisting of neurodegenerative diseases and neurogenic tumors. Pharmaceutical composition for use.