JP4355181B2 - ADIP protein and use thereof - Google Patents

Description

  The present invention relates to a novel afadin DIL domain binding protein (ADIP), a gene encoding the protein, a method for screening a therapeutic agent for a disease using the gene, and a method for diagnosing the disease.

  Cells in multicellular organisms recognize their neighboring cells, adhere to them, and form cell-cell junctions. Such binding has an indispensable role in various cell functions such as morphogenesis, differentiation, proliferation and migration (see Non-Patent Documents 1 to 6). In polarized epithelial cells, cell junctions are mediated by a junction complex consisting of tight junctions (TJs), adhesive junctions (AJs), and desmosomes. These binding structures are usually aligned from the tip to the basal side, but desmosomes are distributed independently in other regions.

AJs were initially defined using ultrastructural analysis as a plasma membrane domain lined up with dense cytoplasmic plaques lined with actin filament (F-actin) bundles (see Non-Patent Document 7). . According to molecular analysis, AJs show that typical cadherins function like cell adhesion molecules and are cell-cell adhesion sites where the actin-based cytoskeleton and several cytoplasmic components assemble (See Non-Patent Documents 8 and 9). E-cadherin, like other typical cadherins, is a single-pass transmembrane protein whose extracellular domain mediates homophilic recognition and adhesive binding in a Ca ²⁺ dependent manner (Non-Patent Document 10). reference). E-cadherin associates with the actin cytoskeleton through surrounding membrane proteins including α-, β- and γ-catenin, α-actinin and vinculin (see Non-Patent Documents 11-14). β-catenin directly interacts with the cytoplasmic tail of E-cadherin to bind E-cadherin to α-catenin that is directly bound to F-actin (see Non-Patent Document 15). α-actinin and vinculin are F-actin-binding proteins that are directly bound to α-catenin (see Non-Patent Documents 13, 14, and 16).

  The association of E-cadherin with the actin cytoskeleton through these peripheral membrane proteins enhances cell-cell adhesion of E-cadherin (see Non-Patent Documents 1 and 17).

The present inventors have found that Nectin, another cell-cell adhesion molecule, and Afadin, an F-actin binding protein associated with Nectin, are also localized in AJs (Non-patent Documents 18 and 19). reference). Nectin and afadin are strictly localized to AJs, using ultrastructural analysis as a closely arranged plasma membrane domain reinforced by dark cytoplasmic plaques lined with F-actin bundles (See Non-Patent Documents 7, 18, and 19). In contrast, E-cadherin is concentrated in Ajs, but is more widely distributed from the tip of the lateral plasma membrane to the basal side (see Non-Patent Documents 2 and 20). Nectin is a Ca ²⁺ independent immunoglobulin-like cell-cell adhesion molecule (see Non-Patent Documents 19, 21 to 25). Nectin currently comprises a family of four membranes, namely Nectin-1, -2, -3, and -4. All Nectins except Nectin-4 have 2 or 3 splice variants and Nectin-1α, -1β, -2α, -2δ, -3α, -3β, and -3γ isoforms are present ( Non-patent document 19, 24-30). Nectin-1 was first identified as one of the poliovirus receptor-related proteins (PRR1) (see Non-Patent Document 29). Nectin-2 was first identified as a murine homologue of human poliovirus receptor protein (see Non-Patent Document 26), but later proved to be another poliovirus receptor-related protein (PRR2) (Non-Patent Document). 28 and 29). They were later shown to act as receptors for α-herpesvirus, facilitating the invasion and intracellular spread of the virus, and were renamed HveC and -B, respectively (Non-Patent Documents 30- 35). All members have an extracellular domain with three immunoglobulin-like loops, a single transmembrane region, and a cytoplasmic region. In addition, all members except Nectin-1β, -3γ, and -4 have a conserved motif of 4 amino acid residues (Glu / Ala-X-Tyr-Val) at its carboxyl terminus. This motif binds to the PDZ domain of afadin (see Non-Patent Documents 18, 19, 24, and 25).

  Afadin has at least two splice variants, namely l-afadin and s-afadin (see Non-Patent Document 18). The larger splice variant, l-afadin, binds to nectin and also binds to F-actin via the F-actin binding domain. l-Afadin binds to the side of F-actin but does not crosslink to form a bundle. Afadin has two Ras association domains (RA), forkhead association domain (FHA), dilution domain (DIL), PDZ domain, two proline-rich domains (PR), and one F-actin binding PR domain ( (See FIG. 1A). DIL domains are found in afadin and type V myosins including dilute (DIL), Myo2 and Myo4. However, its function is still unclear (see Non-Patent Document 36). Recent findings that the Myo4 region containing a DIL domain binds to She3, an adapter protein (see Non-Patent Documents 37 and 38), indicate that the DIL domain is involved in protein-protein interactions.

  The smaller splice variant, s-Afadin, has two RA, FHA, DIL, PDZ, and two PR domains, but lacks the F-actin binding PR domain. Human s-afadin is identical to the gene product of AF-6, which is a gene identified as an ALL-1 fusion pair involved in acute myeloid leukemia (see Non-Patent Document 39). Unless otherwise specified, in the present specification, afadin means l-afadin.

  Nectin has the effect of supplementing E-cadherin-β-catenin complex to the cell-cell adhesion site based on nectin via afadin and α-catenin in fibroblasts and epithelial cells (non-patent literature) 40 and 41). Nectin is also a component of TJ that contains ZO-1, claudin, occludin, and junctional adhesion molecules (JAM) at the cell-cell adhesion site based on nectin via afadin in fibroblasts and epithelial cells. (See Non-Patent Documents 41 to 43). Claudin is an important cell-cell adhesion molecule that forms a TJ chain (see Non-Patent Documents 44 to 47), and occludin and JAM are other transmembrane proteins in TJs (Non-Patent Documents 46 to 48). reference). Claudin, occludin, and JAM interact with ZO-1, which is an F-actin-binding scaffold molecule (see Non-Patent Documents 49-60). In the epithelial cells of afadin (− / −) mice and (− / −) embryoid bodies, proper organization of AJs and TJs is impaired (see Non-Patent Document 61). Nectin-1 has recently been determined by positional cloning to be involved in cleft lip / palate-ectodermal dysplasia characterized by cleft lip / palat, joint finger, mental retardation, and ectoderm dysplasia (Non-Patent Document 62). reference).

Furthermore, the present inventors have recently shown that the Nectin-Afadin system is involved in synapse formation in cooperation with N-cadherin in neurons (see Non-Patent Document 63), and the Nectin-Afadin system is a Sertoli in the testis. It has been found that an important adhesion system in organizing the cell-sperm cell junction is constructed (see Non-Patent Document 64). Thus, nectin and afadin are important for the organization of a wide variety of cell junctions, either with or without known cell adhesion molecules. However, the molecular mechanism by which the Nectin-Afadin system organizes these intercellular junctions remains unclear.
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(5 others), "Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin.", J. Biol. Chem., 2000, Vol.275, p.20520 -20526 Ebnet, K. (4 others), "Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1.", J. Biol. Chem., 2000, Vol. 275, p. 27979 -27988 Wittchen, ES (2 others), "Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly.", J. Cell Biol., 2000, Vol. 151, p.825 -836 Itoh, M. (5 others), "Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions.", J. Cell Biol., 2001, Vol. .154, p.491-497 Ikeda, W. (12 others), “Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.”, J. Cell Biol., 1999, Vol.146, p.1117 -1132 Suzuki, K. 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  The present invention has been made in view of such circumstances, and an object thereof is to provide a novel afadin DIL domain binding protein (ADIP) gene. Another object of the present invention is to provide a novel application of ADIP identified as described above.

  The present inventors have intensively studied to solve the above problems. First, the inventors identified afadin binding proteins by yeast two-hybrid screening to gain insight into how the nectin and afadin systems organize AJs and TJs. Then, using the DIL domain of afadin as a bait, the inventors succeeded in identifying ADIP (afadin DIL domain interacting protein), which is a novel afadin binding protein that binds to the domain.

  ADIP was further found to bind to α-actinin, an F-actin bundle-forming protein known to associate indirectly with E-cadherin via direct binding to α-catenin ( Knudsen, KA, AP Soler, KR Johnson, and MJ Wheelock. 1995. Interaction of alpha-actinin with the cadherin / catenin cell-cell adhesion complex via alpha-catenin. J. Cell Biol. 130: 67-77.). These results suggest that ADIP is connected to the nectin-afadin and E-cadherin-catenin systems through α-actinin and is involved in the organization of the actin cytoskeleton in AJs through afadin and α-actinin. This suggests that ADIP is useful in evaluating drugs that control the actin skeleton.

  Moreover, since ADIP showed very strong expression in the intervening plate of cardiomyocytes, it may be effective as a function marker of the intervening plate in heart diseases such as myocardial infarction and myocarditis.

That is, the present invention relates to a novel ADIP gene and use thereof, more specifically,
[1] The polynucleotide according to any one of (a) to (d) below:
(a) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4
(b) a polynucleotide comprising the coding region of the nucleotide sequence in SEQ ID NO: 1 or 3
(c) a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 2 or 4, and having a binding activity to afadin or actinin A polynucleotide comprising a base sequence encoding
(d) a polynucleotide that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3 and that encodes a protein having a binding activity to afadin or actinin [2 A polypeptide encoded by the polynucleotide according to [1],
[3] A vector into which the polynucleotide according to [1] is inserted,
[4] A host cell carrying the polynucleotide according to [1] or the vector according to [3],
[5] A method for producing the polypeptide according to [2], comprising culturing the host cell according to [4] and recovering the produced polypeptide from the host cell or a culture supernatant thereof,
[6] A polynucleotide that specifically hybridizes with the polynucleotide of [1] under stringent conditions, wherein the polynucleotide has a chain length of at least 15 nucleotides,
[7] An antisense polynucleotide to the polynucleotide according to [1] or a part thereof,
[8] an antibody that binds to the polypeptide of [2],
[9] A method for screening candidate compounds for a drug that regulates the actin skeleton, comprising the following steps (a) to (c):
(a) contacting afadin or actinin with the polypeptide according to [2] and a test compound;
(b) measuring the binding activity between afadin or actinin and the polypeptide according to [2],
(c) A step of selecting a compound that changes the binding activity as compared with the case where the test compound is not contacted [10] Detecting the expression level of the gene encoding the polypeptide according to [2] in the subject An examination method for heart disease,
[11] The inspection method according to [10], including the following steps (a) to (c):
(A) a step of preparing an RNA sample from a subject's cardiomyocytes (b) a step of measuring the amount of RNA encoding the polypeptide according to [2] contained in the RNA sample (c) the measured RNA In a preferred embodiment of the above step, when the amount of RNA encoding the polypeptide of the present invention is significantly changed as compared with the control, the subject Determining that the patient is suffering from, or at risk of developing, a heart disease associated with abnormal expression,
[12] The inspection method according to [10], including the following steps (a) to (c):
(A) a step of preparing a protein sample from a subject's cardiomyocytes (b) a step of measuring the amount of the polypeptide according to [2] contained in the protein sample (c) an amount of the measured polypeptide Step of comparing with control In a preferred embodiment of the above step, when the amount of the polypeptide of the present invention is significantly changed compared to control, the subject is considered to be associated with abnormal expression of the gene. Determining that the patient has a heart disease or is at risk of developing the disease,
[13] The examination method according to any one of [10] to [12], wherein the heart disease is myocardial infarction or myocarditis,
[14] The polynucleotide according to [1], wherein the polynucleotide is (a),
[15] The polynucleotide according to [1], wherein the polynucleotide is (b),
[16] The polynucleotide (c) is a polynucleotide comprising an amino acid sequence in which 10% or less of amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2 or 4. The polynucleotide according to [1],
[17] The polynucleotide of [1], wherein the polynucleotide of (c) is a polynucleotide encoding a protein having at least 70% homology with the amino acid sequence of SEQ ID NO: 2 or 4.
[18] The polynucleotide of [1], wherein the polynucleotide of (d) is a polynucleotide having at least 70% homology with the base sequence of SEQ ID NO: 1 or 3.
[19] The polypeptide of [2], which is a polypeptide having at least 70% homology with the amino acid sequence of SEQ ID NO: 2 or 4.
[20] The polypeptide according to [2], comprising the amino acid sequence according to SEQ ID NO: 2 or 4.

  The present inventors have provided a novel afadin DIL domain binding protein (ADIP) and a gene encoding the protein. The protein of the present invention is thought to be useful in evaluating drugs that control the actin skeleton.

  Further, since the protein of the present invention shows very strong expression in the intervening plate of cardiomyocytes, it is effective as a functional marker of the intervening plate in heart diseases such as myocardial infarction and myocarditis. By using the expression level of the gene of the present invention as an index, a heart disease can be diagnosed. In addition, the protein of the present invention is useful for screening drug candidate compounds for treating heart diseases.

  The present invention provides a polynucleotide encoding ADIP, a novel protein that binds to the afadin DIL domain. The nucleotide sequence of the polynucleotide encoding mouse ADIP identified by the present inventors, which is included in the present invention, is shown in SEQ ID NO: 1, and the amino acid sequence of the polypeptide encoded by the polynucleotide is shown in SEQ ID NO: 2. . The nucleotide sequence of a polynucleotide encoding rat ADIP identified by the present inventors, which is included in the present invention, is shown in SEQ ID NO: 3, and the amino acid sequence of the polypeptide encoded by the polynucleotide is shown in SEQ ID NO: 4. Show.

  The present invention also relates to polypeptides functionally equivalent to the polypeptides identified by the inventors, and polynucleotides encoding the polypeptides. Here, “functionally equivalent” means that the polypeptide of interest has biological properties equivalent to the polypeptide identified by the present inventors. Examples of biological properties possessed by ADIP include activity to bind to afadin and / or actinin. Accordingly, the present invention includes an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 2 or 4, and has a binding activity to afadin or actinin. A polynucleotide comprising a base sequence encoding a protein having Furthermore, a polynucleotide that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3 and that encodes a protein having binding activity to afadin or actinin is also included in the present invention. include. The measurement of the “binding activity” can be carried out by those skilled in the art, for example, a yeast two-hybrid system. In the present invention, “actinin” usually refers to “α-actinin”.

  The polynucleotide of the present invention can be isolated by those skilled in the art by generally known methods. For example, hybridization techniques (Southern, EM., J Mol Biol, 1975, 98, 503.) and polymerase chain reaction (PCR) techniques (Saiki, RK. Et al., Science, 1985, 230, 1350., Saiki, RK. Et al., Science 1988, 239, 487.). That is, an oligonucleotide that specifically hybridizes to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3 or a part thereof as a probe, or to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3 It is usually possible for those skilled in the art to isolate a polynucleotide having a high homology with a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or 3 from other animals (eg, human etc.) using as a primer That is. Thus, the polynucleotide which hybridizes with the polynucleotide which consists of a base sequence of sequence number 1 or 3 which can be isolated by a hybridization technique or PCR technique is also contained in the polynucleotide of this invention. Examples of such a polynucleotide include a polynucleotide encoding a human homologue of ADIP.

  In order to isolate the polynucleotide, a hybridization reaction is preferably performed under stringent conditions. In the present invention, stringent hybridization conditions refer to hybridization conditions of 6M urea, 0.4% SDS, 0.5 × SSC or equivalent stringency. It is expected that polynucleotides with higher homology can be isolated under conditions of higher stringency, for example, 6M urea, 0.4% SDS, 0.1 × SSC. The isolated polynucleotide is considered to have high homology with the amino acid sequence shown in SEQ ID NO: 2 or 4 at the amino acid level. High homology refers to sequence identity of at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more of the entire amino acid sequence.

  The identity of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 1990, 87, 2264-2268., Karlin, S. & Altschul, SF., Proc. Natl by Carlin and Arthur. Acad. Sci. USA, 1993, 90, 5873.). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul, SF. Et al., J Mol Biol, 1990, 215, 403.). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov/).

  The polynucleotide of the present invention includes genomic DNA, cDNA and RNA, as well as chemically synthesized DNA and RNA. The chain length of the polynucleotide of the present invention is not particularly limited, but is at least 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 1000, 1500, 2000, 2500, or 3000 nucleotides. A chain length is preferred. Genomic DNA and cDNA can be prepared by those skilled in the art by conventional means. For example, genomic DNA is extracted from an organism having a polynucleotide encoding ADIP, and a genomic library (for example, plasmids, phages, cosmids, BACs, PACs, etc. can be used as vectors), By developing this, colony hybridization or plaque hybridization can be performed using a probe prepared based on a polynucleotide encoding ADIP of the present invention (for example, SEQ ID NO: 1 or 3). It is also possible to prepare a primer specific for a polynucleotide encoding ADIP of the present invention (for example, SEQ ID NO: 1 or 3) and perform PCR using this primer. For example, cDNA is synthesized based on mRNA extracted from an organism having a polynucleotide encoding ADIP, and inserted into a vector such as λZAP to create a cDNA library. It can be prepared by performing colony hybridization or plaque hybridization in the same manner as described above, or by performing PCR.

  The present invention also provides a polynucleotide encoding a protein that is structurally similar to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4. Examples of such a polynucleotide include a polynucleotide comprising a base sequence encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the protein. The number of amino acid mutations and mutation sites in the above protein are the functions (for example, functions described in the following literature: Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, MJ & Smith, M., Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413) is not limited. The number of mutations will typically be within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids.

  The amino acid residue into which the mutation is introduced is preferably an amino acid that retains the properties of the amino acid before the mutation and the side chain (conservative substitution of amino acids). Examples of the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), and hydrophilicity (R, D, N, C, E, Q, G). , H, K, S, T). Moreover, as an amino acid side chain, the following functions and characteristics can be exemplified, for example. Aliphatic amino acids (G, A, V, L, I, P), hydroxyl amino acids (S, T, Y), sulfur amino acids (C, M), carboxy amino acids and amide amino acids (D, N, E, Q) , Basic amino acids (R, K, H), aromatic amino acids (H, F, Y, W).

  Examples of proteins to which one or more amino acids are added include fusion proteins including ADIP proteins. The fusion protein of the present invention can be appropriately prepared by methods well known to those skilled in the art. For example, there is a method of binding DNA encoding the ADIP protein of the present invention and DNA encoding another protein so that the reading frame matches, but this method is not particularly limited. As a peptide to be fused with the protein of the present invention, various known peptides can be used. For example, FLAG (Hopp, TP et al., Biotechnology (1988) 6, 1204-1210), 6x His, 10x His, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIV Examples include fragments, T7-tag, HSV-tag, E-tag, SV40T antibody fragment, lck tag, α-tubulin fragment, B-tag, protein C fragment and the like. Examples of proteins to be fused with the protein of the present invention include GST (glutathione-S-transferase), influenza agglutinin, immunoglobulin constant region, β-galactosidase, MBP (maltose binding protein) and the like.

  In order to prepare the polynucleotide, methods well known to those skilled in the art include, for example, the site-directed mutagenesis method for the polynucleotide in addition to the hybridization technique and the polymerase chain reaction (PCR) technique. (Kramer, W. & Fritz, HJ., Methods Enzymol, 1987, 154, 350.). Also in nature, it is possible that the amino acid sequence of the encoded protein is mutated due to the mutation of the base sequence. In addition, even if the base sequence is mutated, there are cases where the mutation does not accompany amino acid mutation (degenerate mutation), and such degenerate mutant polynucleotides are also included in the present invention. In addition, a polypeptide encoded by the polynucleotide of the present invention is also included in the present invention.

  The present invention provides a vector into which the polynucleotide of the present invention is inserted, the polynucleotide of the present invention or a host cell holding the vector, and a method for producing the polypeptide of the present invention using the host cell.

  The vector of the present invention is not particularly limited as long as it can stably hold the inserted DNA. For example, if Escherichia coli is used as the host, the cloning vector is preferably a pBluescript vector (Stratagene). An expression vector is particularly useful when a vector is used for the purpose of producing the polypeptide of the present invention. The expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in vitro, in E. coli, in cultured cells, or in an individual organism. For example, in the case of in vitro expression, pBEST vector (manufactured by Promega), E. coli PET vector (manufactured by Invitrogen), pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, pME18S vector (Mol Cell Biol. 8: 466-472 (1988)) for organisms, etc. Is preferred. The insertion of the DNA of the present invention into a vector can be performed by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons, Section 11.4-11.11).

  The host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells can be used depending on the purpose. Examples of cells for expressing the polypeptide include bacterial cells (eg, Streptococcus, Staphylococcus, E. coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), insect cells (eg, Drosophila S2). , Spodoptera SF9), animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells. Vector introduction into host cells can be performed by, for example, calcium phosphate precipitation, electric pulse perforation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofectamine method (GIBCO -BRL) and a known method such as a microinjection method.

  In order to secrete the polypeptide expressed in the host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, an appropriate secretion signal can be incorporated into the polypeptide of interest. These signals may be endogenous to the polypeptide of interest or may be heterologous signals.

  The polypeptide of the present invention is recovered when the polypeptide of the present invention is secreted into the medium. When the polypeptide of the present invention is produced intracellularly, the cell is first lysed, and then the polypeptide is recovered.

  To recover and purify the polypeptides of the invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, Known methods including hydroxylapatite chromatography and lectin chromatography can be used.

  The present invention provides a nucleotide having a chain length of at least 15 nucleotides, which is complementary to the polynucleotide identified by the present inventors (polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 3 or its complementary strand). provide. Here, “complementary strand” refers to the other strand with respect to one strand of a double-stranded nucleic acid consisting of A: T (U in the case of RNA) and G: C base pairs. Further, the term “complementary” is not limited to a case where the sequence is completely complementary in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95%. What is necessary is just to have the homology on the above base sequences. The algorithm described in the present specification may be used as an algorithm for determining homology. Such a nucleotide can be used as a probe for detecting and isolating the polynucleotide of the present invention and as a primer for amplifying the nucleotide of the present invention. When used as a primer, it usually has a chain length of 15 to 100 nucleotides, preferably 15 to 35 nucleotides. When used as a probe, a polynucleotide having a chain length of at least 15 nucleotides, preferably at least 30 nucleotides, containing at least a part or all of the sequence of the DNA of the present invention is used. Such a polynucleotide preferably hybridizes specifically to a polynucleotide encoding the polypeptide of the present invention. “Specifically hybridize” means to hybridize with the polynucleotide (SEQ ID NO: 1 or 3) identified by the present inventors under normal hybridization conditions, preferably under stringent conditions, This means that it does not hybridize with DNA encoding another polypeptide.

  In addition, the polynucleotide includes a polynucleotide that suppresses the expression of a gene encoding the polypeptide of the present invention. Such polynucleotides include antisense polynucleotides (antisense DNA / RNA; antisense RNA complementary to the transcript of the gene encoding the polypeptide of the present invention, and DNA encoding the RNA) and ribozymes ( DNA encoding an RNA having ribozyme activity that specifically cleaves the transcript of the gene encoding the polypeptide of the present invention.

  There are several factors as described below for the action of an antisense polynucleotide to suppress the expression of a target gene. That is, transcription initiation inhibition by triplex formation, transcription inhibition by hybridization with a site where an open loop structure is locally created by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, intron and exon Of splicing by hybrid formation at the junction with the protein, suppression of splicing by hybridization with the spliceosome formation site, suppression of transition from the nucleus to the cytoplasm by hybridization with mRNA, hybridization with capping sites and poly (A) addition sites Splicing suppression by formation, translation initiation suppression by hybridization with the translation initiation factor binding site, translation suppression by hybridization with the ribosome binding site near the initiation codon, peptization by hybridization with the mRNA translation region and polysome binding site For example, inhibition of gene chain elongation is prevented, and hybridization between nucleic acid and protein interaction sites is performed. They inhibit transcription, splicing, or translation processes and suppress target gene expression (Hirashima and Inoue, "Neurochemistry Experiment Course 2, Nucleic Acid IV Gene Replication and Expression", edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin , pp.319-347, 1993).

  The antisense polynucleotide used in the present invention may suppress the expression of the target gene by any of the actions described above. As one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the gene is designed, it is considered effective for inhibiting the translation of the gene. However, sequences complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, a polynucleotide containing an antisense sequence of not only a translation region of a gene but also a sequence of an untranslated region is also included in the antisense polynucleotide used in the present invention. The antisense polynucleotide to be used is linked downstream of an appropriate promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The antisense polynucleotide sequence is preferably a sequence that is complementary to the target gene or a part thereof, but may not be completely complementary as long as it can effectively inhibit the expression of the gene. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene. In order to effectively inhibit the expression of the target gene using the antisense sequence, the antisense polynucleotide is at least 15 nucleotides, preferably 100 nucleotides, more preferably 500 nucleotides or more, in order to cause an antisense effect. The chain length is usually within 3000 nucleotides, preferably within 2000 nucleotides.

  The antisense polynucleotide is, for example, based on the sequence information of a polynucleotide (eg, SEQ ID NO: 1 or 3) encoding the polypeptide of the present invention (Stein, 1988 Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 16, 3209-21 (1988)).

  Suppression of endogenous gene expression can also be performed using a polynucleotide encoding a ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities. Among them, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for site-specific cleavage of RNA. Some ribozymes have a group I intron type and a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, (1990) Protein Nucleic Acid Enzymes, 35: 2191).

  For example, the self-cleaving domain of hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for U14 to form a base pair with A at position 9, and the base at position 15 is In addition to C, it has been shown to be cleaved by A or U (M. Koizumi et al., (1988) FEBS Lett. 228: 225). If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. (M.Koizumi et al., (1988) FEBS Lett. 239: 285, Makoto Koizumi and Eiko Otsuka, (1990) Protein Nucleic Acid Enzymes, 35: 2191, M. Koizumi et al., (1989) Nucleic Acids Res. 17: 7059 ).

  Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of tobacco ring spot virus satellite RNA (J. M. Buzayan Nature 323: 349, 1986). It has been shown that this ribozyme can also be designed to cause target-specific RNA cleavage (Y. Kikuchi and N. Sasaki (1992) Nucleic Acids Res. 19: 6751, Hiroshi Kikuchi, (1992) Chemistry and Biology 30 : 112).

  When the polynucleotide that suppresses the expression of the gene encoding the polypeptide of the present invention is used for gene therapy, for example, a viral vector such as a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a non-viral such as a liposome. It is conceivable to administer the patient by ex vivo method or in vivo method using a vector or the like.

  The present invention provides antibodies that bind to the polypeptides of the present invention. As used herein, “antibodies” include polyclonal and monoclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, and Fab fragments including the products of Fab or other immunoglobulin expression libraries.

  The polypeptides of the invention or fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies that bind to the polypeptides of the invention. The antibody is preferably immunospecific for a polypeptide of the invention. “Immunospecific” means that the antibody has a substantially higher affinity for a polypeptide of the invention than its affinity for another polypeptide.

  Antibodies that bind to the polypeptides of the present invention can be prepared by methods known to those skilled in the art. If it is a polyclonal antibody, it can obtain as follows, for example. Serum is obtained by immunizing a small animal such as a rabbit with the polypeptide of the present invention or a fusion protein thereof with GST. This is prepared, for example, by purifying with ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with the polypeptide of the present invention, and the like. In the case of a monoclonal antibody, for example, the polypeptide of the present invention is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate cells, such as mouse myeloma cells and polyethylene glycol. A clone that produces an antibody that binds to the polypeptide of the present invention is selected from the fused cells (hybridoma) formed by fusion with a reagent. Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, It can be prepared by purification using an affinity column coupled with a polypeptide.

  The antibody of the present invention can be used for isolation, identification, and purification of the polypeptide of the present invention and cells expressing the polypeptide.

  The polypeptides of the present invention can be used in the screening of candidate compounds for agents that control the actin backbone. An agent that regulates the actin skeleton can be a therapeutic agent for a disease associated with abnormal expression (function) of the polypeptide of the present invention. The molecule to be identified may be of natural origin or an artificially synthesized structural or functional mimetic. The polypeptides of the present invention are believed to be involved in many biological functions, including many pathologies. Accordingly, it is desirable to discover compounds that activate the polypeptides of the present invention and compounds that can inhibit activation of the polypeptides of the present invention.

  The present invention provides methods for screening candidate compounds for agents that control the actin skeleton.

  In this method, first, afadin or actinin is brought into contact with the polypeptide of the present invention and a candidate compound, and then the binding activity between afadin or actinin and the polypeptide of the present invention is measured. Then, a compound that changes (increases or suppresses) the binding activity is selected as compared with the case where the test compound is not contacted.

  The compound isolated by the above screening method of the present invention as a compound that changes the binding activity of the polypeptide of the present invention to afadin or actinin is a therapeutic agent for heart diseases such as myocardial infarction and myocarditis. Be expected. Moreover, this compound can also be used for the said screening method of this invention as a test compound.

  The measurement of the binding activity can be appropriately carried out by those skilled in the art by a method well known, for example, a yeast two-hybrid system. The test compound is not particularly limited. For example, various known compounds and peptides (for example, those registered in a chemical file) or phage display method (J. Mol. Biol. (1991) 222, 301- 310) or the like can be used to create a random peptide group. In addition, culture supernatants of microorganisms, natural components derived from plants and marine organisms, and the like are also subject to screening. Other examples include biological tissue extracts, cell extracts, and expression products of gene libraries, but are not particularly limited thereto.

  The present invention also relates to a method for examining a disease associated with abnormal expression of a gene encoding the polypeptide of the present invention. The polypeptide of the present invention is considered to have an important function in vivo, and abnormal expression thereof can cause various diseases. Therefore, it is possible to examine such diseases by using the expression of the polypeptide of the present invention as an index.

  In the present invention, “disease test” is performed not only for a test for establishing a treatment strategy for a subject exhibiting a symptom of a disease, but also for determining whether or not the subject is susceptible to the disease. Also included are tests for prevention, or tests for whether or not already affected.

  Since ADIP is very strongly expressed in the intervening plate of cardiomyocytes, it is possible that abnormal expression of ADIP may cause heart disease. Therefore, the disease of the present invention is usually a heart disease, and more specifically, myocardial infarction or myocarditis can be exemplified.

  One embodiment of the test method of the present invention is a method comprising detecting a mutation in a gene encoding the polypeptide of the present invention or an expression control region thereof in a subject.

  One method is a method in which a test is performed by directly determining the base sequence of the gene encoding the polypeptide of the present invention or the expression control region thereof in a subject. In this method, first, a DNA sample is prepared from a subject. The DNA sample can be prepared based on chromosomal DNA or RNA extracted from a subject's cells, for example, cardiomyocytes. In order to prepare a DNA sample of the present method from chromosomal DNA, for example, chromosomal DNA may be cleaved with an appropriate restriction enzyme and cloned into a vector to prepare a genomic library. In order to prepare a DNA sample of the present method from RNA, for example, a cDNA library may be prepared from RNA using reverse transcriptase. In this method, next, a DNA encoding a gene encoding the polypeptide of the present invention or an expression control region thereof is isolated. The DNA can be isolated by screening a genomic library or cDNA library using a probe that hybridizes to the gene encoding the polypeptide of the present invention or a DNA containing the expression control region thereof. . It can also be isolated by genomic DNA library, cDNA library, or PCR using RNA as a template, using a primer that hybridizes to the gene encoding the polypeptide of the present invention or DNA containing its expression control region. it can. In this method, the base sequence of the isolated DNA is then determined. The base sequence of the selected DNA can be determined by a method known to those skilled in the art. In the present method, the determined DNA base sequence is then compared with a control. The “control” in the present method refers to a base sequence of a DNA containing a normal (wild type) gene encoding the polypeptide of the present invention or an expression control region thereof. As a result of such comparison, if the base sequence of the subject's DNA is different from that of the control, the subject is determined to be suffering from or at risk of developing the disease.

  In addition, for example, a base sequence of a gene encoding the protein of the present invention obtained from a patient with a heart disease such as cardiomyopathy or myocardial infarction can be used as the “control” of the present invention. In this case, when the DNA base sequence of the subject is the same as that of the control, it is determined that the subject is suffering from or at risk of developing the disease.

  In the test method of the present invention, various methods can be used in addition to the method for directly determining the base sequence of DNA derived from a subject as described above.

  In one method, a DNA sample is first prepared from a subject. Next, the prepared DNA sample is cleaved with a restriction enzyme. The DNA fragments are then separated according to their size. The size of the detected DNA fragment is then compared to a control. In another embodiment, a DNA sample is first prepared from a subject. Next, a gene encoding the polypeptide of the present invention or a DNA containing its expression control region is amplified. Furthermore, the amplified DNA is cleaved with a restriction enzyme. The DNA fragments are then separated according to their size. The size of the detected DNA fragment is then compared to a control.

  Examples of such a method include a method utilizing restriction fragment length polymorphism (RFLP) and a PCR-RFLP method. Specifically, if there is a mutation at the restriction enzyme recognition site, or if there is a base insertion or deletion in the DNA fragment produced by the restriction enzyme treatment, the size of the fragment produced after the restriction enzyme treatment is compared with the control. Change. By amplifying a portion containing this mutation by PCR and treating with each restriction enzyme, these mutations can be detected as a difference in mobility of bands after electrophoresis. Alternatively, the presence or absence of mutation can be detected by treating chromosomal DNA with these restriction enzymes, performing electrophoresis, and performing Southern blotting using the probe DNA of the present invention. The restriction enzyme used can be appropriately selected according to each mutation. In this method, in addition to genomic DNA, RNA prepared from a subject can be converted to cDNA using reverse transcriptase, cut with a restriction enzyme as it is, and then subjected to Southern blotting. It is also possible to amplify a DNA encoding the polypeptide of the present invention or a DNA containing its expression control region by PCR using this cDNA as a template, cleave it with a restriction enzyme, and then examine the difference in mobility. .

  In another method, a DNA sample is first prepared from a subject. Next, a gene encoding the polypeptide of the present invention or a DNA containing its expression control region is amplified. Furthermore, the amplified DNA is dissociated into single-stranded DNA. The dissociated single-stranded DNA is then separated on a non-denaturing gel. The mobility of the separated single-stranded DNA on the gel is compared with the control.

  Such methods include, for example, the PCR-SSCP (single-strand conformation polymorphism) method (Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11.Genomics 1992 Jan 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene. 1991 Aug 1; 6 (8): 1313- 1318.). This method is suitable for screening a large number of DNA samples in particular because it is relatively easy to operate and has advantages such as a small amount of test sample. The principle is as follows. When a double-stranded DNA fragment is dissociated into single strands, each strand forms a unique higher-order structure depending on its base sequence. When this dissociated DNA strand is electrophoresed in a polyacrylamide gel containing no denaturing agent, single-stranded DNA having the same complementary strand length moves to a different position according to the difference in each higher-order structure. The higher-order structure of this single-stranded DNA also changes by substitution of a single base, and shows different mobility in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, it is possible to detect the presence of mutations due to point mutations, deletions or insertions in the DNA fragment.

Specifically, first, a DNA encoding a polypeptide encoding the polypeptide of the present invention or an expression control region thereof is amplified by a PCR method or the like. As a range to be amplified, a length of about 200 to 400 bp is usually preferable. Those skilled in the art can perform PCR by appropriately selecting reaction conditions and the like. During PCR, the amplified DNA product can be labeled by using a primer labeled with an isotope such as ³² P, a fluorescent dye, or biotin. Alternatively, the amplified DNA product can be labeled by adding a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin to the PCR reaction solution and performing PCR. Furthermore, labeling can also be performed by adding a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin to the amplified DNA fragment using Klenow enzyme or the like after the PCR reaction. The labeled DNA fragment thus obtained is denatured by applying heat or the like and electrophoresed on a polyacrylamide gel containing no denaturing agent such as urea. At this time, conditions for separating DNA fragments can be improved by adding an appropriate amount (about 5 to 10%) of glycerol to the polyacrylamide gel. Electrophoretic conditions vary depending on the nature of each DNA fragment, but are usually performed at room temperature (20 to 25 ° C). If favorable separation cannot be obtained, the temperature at which the optimal mobility is obtained at temperatures from 4 to 30 ° C. Review. After electrophoresis, the mobility of the DNA fragments is detected and analyzed by autoradiography using an X-ray film, a scanner that detects fluorescence, or the like. When a band with a difference in mobility is detected, this band can be directly excised from the gel, amplified again by PCR, and directly sequenced to confirm the presence of the mutation. Even when the labeled DNA is not used, the band can be detected by staining the gel after electrophoresis with ethidium bromide or silver staining.

  In yet another method, a DNA sample is first prepared from a subject. Next, a gene encoding the polypeptide of the present invention or a DNA containing its expression control region is amplified. Furthermore, the amplified DNA is separated on a gel where the concentration of the DNA denaturant is gradually increased. The mobility of the separated DNA on the gel is then compared to the control.

  Examples of such a method include denaturant gradient gel electrophoresis (DGGE method). The DGGE method is a method in which a mixture of DNA fragments is run in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated depending on the instability of each. When a mismatched unstable DNA fragment migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to its instability. The mobility of this partially dissociated DNA fragment becomes very slow and can be separated from the mobility of a complete double-stranded DNA without a dissociated portion. Specifically, a gene encoding the polypeptide of the present invention or a DNA containing its expression control region is amplified by a PCR method using the primer of the present invention, and the concentration of denaturing agents such as urea is transferred. Electrophorese in a polyacrylamide gel that gradually increases according to and compare to the control. In the case of a DNA fragment with mutation, the DNA fragment becomes single-stranded at a lower denaturant concentration position, and the movement speed becomes extremely slow. Therefore, the presence of mutation is detected by detecting this difference in mobility. be able to.

  In addition to the above method, an allele specific oligonucleotide (ASO) hybridization method can be used for the purpose of detecting only a mutation at a specific position. When an oligonucleotide containing a base sequence that is considered to have a mutation is prepared and hybridized with the sample DNA, the efficiency of hybridization is reduced when the mutation exists. It can be detected by Southern blotting, a method using the property of quenching by intercalating a special fluorescent reagent into the hybrid gap, or the like. Moreover, detection by the ribonuclease A mismatch cleavage method is also possible. Specifically, DNA containing a gene encoding the polypeptide of the present invention is amplified by PCR or the like, and hybridized with labeled RNA prepared from control cDNA or the like incorporated into a plasmid vector or the like. Since the hybrid has a single-stranded structure in the portion where the mutation exists, the presence of the mutation can be detected by cleaving this portion with ribonuclease A and detecting this by autoradiography or the like.

  Another embodiment of the test method of the present invention is a method comprising detecting the expression level of a gene encoding the polypeptide of the present invention. Herein, “gene expression” includes transcription and translation. Thus, “expression product” includes mRNA and protein.

  In a preferred embodiment of the present invention, there is provided a method for examining a heart disease, which comprises detecting the expression level of a gene encoding the polypeptide of the present invention in a subject.

  In this method, an RNA sample is first prepared from a subject's cells (for example, cardiomyocytes), and the amount of RNA encoding the polypeptide of the present invention in the sample is measured. Then, the measured RNA amount is compared with the control.

  Such methods include Northern blotting using a probe that hybridizes to a polynucleotide encoding the polypeptide of the present invention, or RT- using a primer that hybridizes to a polynucleotide encoding the polypeptide of the present invention. The PCR method etc. can be illustrated.

  Further, in the examination at the transcription level of the gene encoding the polypeptide of the present invention, a DNA array (New Genetic Engineering Handbook, Masaaki Muramatsu / Masaka Yamamoto, Yodosha, p280-284) can be used. Specifically, first, a cDNA sample prepared from a subject and a substrate on which a polynucleotide probe that hybridizes with a polynucleotide encoding the polypeptide of the present invention is immobilized are provided. A plurality of types of polynucleotide probes may be immobilized on the substrate in order to detect a plurality of types of polynucleotides encoding the polypeptides of the present invention. Preparation of a cDNA sample from a subject can be performed by methods well known to those skilled in the art. In a preferred embodiment of preparing a cDNA sample, first, total RNA is extracted from a subject's cells. Examples of the cells include cardiomyocytes. Extraction of total RNA can be performed, for example, as follows. Existing methods and kits can be used as long as they can prepare high-purity total RNA. Next, cDNA is synthesized using the extracted total RNA as a template and reverse transcriptase to prepare a cDNA sample. Synthesis of cDNA from total RNA can be performed by methods well known to those skilled in the art. The prepared cDNA sample is labeled for detection as necessary. The labeling substance is not particularly limited as long as it can be detected, and examples thereof include fluorescent substances and radioactive elements. The labeling can be performed by a method generally performed by those skilled in the art (L Luo et al., Gene expression profiles of laser-capture dad jacent neuronal subtypes. Nat Med. 1999, 117-122).

  The term “control” as used herein usually refers to the amount of RNA encoding the polypeptide of the present invention in an RNA sample prepared from cardiomyocytes of a healthy person.

  In the above method, when the amount of RNA encoding the polypeptide of the present invention is significantly changed compared to the control, the subject suffers from a heart disease that is associated with abnormal expression of the gene. Or is at risk of developing the disease. In addition, in a subject who has already been found to have heart disease, the cause is determined to be a change in the expression level of RNA encoding the polypeptide.

  In addition, the test method of the present invention can be carried out as follows by measuring the protein expression level in the subject's cells (for example, cardiomyocytes). First, a protein sample is prepared from a subject's cells. Next, the amount of the polypeptide of the present invention contained in the protein sample is measured. The measured amount of the polypeptide is then compared to a control.

  Such methods include SDS polyacrylamide electrophoresis and Western blotting, dot blotting, immunoprecipitation, enzyme-linked immunoassay (ELISA), and immunofluorescence using antibodies that bind to the protein. Can be illustrated.

  In the above method, when the expression level of the protein encoded by the polynucleotide of the present invention is significantly changed compared to the control, the subject is suspected of developing heart disease in the future (risk It is determined that the patient is already suffering from heart disease. Further, in a subject who has already been found to have a heart disease, the cause is determined to be a change in the expression level of the polynucleotide described above.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[Example 1] Expression vector construction The mammalian expression vectors pCMV-FLAG, pCMV-T7, and pCMV-HA were designed to express N-terminal FLAG-, T7-, and HA-tagged proteins, respectively. (Takaesu, G., S. Kishida, A. Hiyama, K. Yamaguchi, H. Shibuya, K. Irie ,. J. Ninomiya-Tsuji, and K. Matsumoto. 2000. TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signaltransduction pathway. Mol. Cell. 5: 649-658.).

  A mammalian expression vector expressing afadin DIL domain (aa606 to 983) was constructed using pCMV-T7.

  Mammalian expression vectors expressing mouse ADIP (mADIP) and rat ADIP (rADIP), pCMV-HA-mADIP (aa1-615), pCMV-HA-rADIP-C (aa159-613), pCMV-HA -mADIP-C (aa339-615), pCMV-Flag-mADIP-C (aa339-615), and pCMV-Flag-mADIP-M (aa152-436) were constructed using pCMV-FLAG and pCMV-HA .

  pCMV-HA-α-actinin-1-C (aa406-892), a mammalian expression vector expressing α-actinin-1 (human, BC015766; GenBank), was constructed using pCMV-HA.

  mADIP and rADIP glutathione S-transferase (GST) fusion or MBP-fusion vectors MBP-mADIP (aa1 to 615), MBP-rADIP-C (aa159 to 613), MBP-mADIP-C (aa339 to 615), GST-rADIP-C (aa159 to 613), GST-mADIP-N (aa1 to 226) and GST-mADIP-C (aa339 to 615) are pGEX-KG (Guan, KL, and JE Dixon. 1991. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192: 262-267.) and pMal-C2 (New England Biolabs).

[Example 2] Preparation of antibody
Each GST fusion protein having a fragment of rADIP C-terminal part (aa159 to 613; rADIP), mADIP N-terminal part (aa1 to 226; mADIP-N) and mADIP C-terminal part (aa339 to 615; mADIP-C) Was used as an antigen to raise rabbit pAb after preparation and purification in E. coli.

  Two types of pAbs for rADIP and mADIP-C, M05 and M01, were used after affinity purification with MBP-rADIP-C (aa159-613) and MBP-mADIP-C (aa339-615), respectively. M57, a pAb against mADIP-N, was used after affinity purification with GST-mADIP-N (aa1-226).

  GST- and MBP-fusion proteins were purified using glutathione-Sepharose beads (Amersham-Pharmacia Biotech) and amylose resin beads (New England Biolabs), respectively.

  Mouse anti-Afadin mAb was prepared as described in the literature (Sakisaka, T, H. Nakanishi, K. Takahashi, K. Mandai, M. Miyahara, A. Satoh, K. Takaishi, and Y. Takai. 1999. Different behavior of l-afadin and neurabin-II during the formation and destruction of cell-cell adherens junction. Oncogene. 18: 1609-1617.).

  Rat anti-E-cadherin mAb (ECCD2) is Donated by Dr. Takeichi (Center for Developmental Biology, RIKEN, Kobe, Japan) (A. Nagafuchi, Y. Shirayoshi, K. Okazaki, K. Yasuda, and M. Takeichi, "Transformation of cell adhesion properties by exogenously introduced E -cadherin cDNA. ", Nature, 1987, 329 (6137): 341-3) and Y. Shirayoshi et al. (Y. Shirayoshi, TS Okada, and M. Takeichi," The calcium-dependent cell-cell adhesion system regulates inner cell mass formation and cell surface polarization in early mouse development. ", Cell, 1983, 35 (3 Pt 2): 631-8.). Mouse anti-ZO-1 mAb was purchased from Chemicon. Mouse anti-α-actinin, anti-vinclin and anti-Flag-M2 mAb were purchased from Sigma Chemicals. Rabbit anti-α-actin pAb was purchased from Santa Cruz. Mouse anti-T7 mAb was purchased from Novagen.

[Example 3] Cell culture and protein concentration
HEK293, MDCK, Nectin-2α-L and Nectin-2α-ΔC-L cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum.

  Protein concentration was determined using bovine serum albumin as a reference protein (Bradford, MM 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248 -254.)

Example 4 Identification of Afadin Binding Proteins To identify proteins that bind to afadin, we used the region of afadin containing the DIL domain (aa606-983) as a bait, Hybrid screening was performed. A schematic structure of afadin is shown in FIG. 1A.

  PGBDU-Afadin (aa511 to 981), a bait vector, inserts an aphadine aa residue into pGBDU-C1 (James, P., J. Hallady, and EA Craig. 1996. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 144: 1425-1436).

  A yeast two-hybrid library constructed from 11 day old mouse embryos, rat brain, rat lung, and human testis cDNA was purchased from Clontech.

  Publication of two-hybrid screening using yeast strain PJ69-4A (MATa trp1-901 leu2-3, 112 ura3-52 his3-200 gal4Δ gal80Δ GAL2-ADE2 LYS2 :: GAL1-HIS3 met2 :: GAL7-lacZ) (James , P., J. Hallady, and EA Craig. 1996. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 144: 1425-1436). Yeast manipulation was performed as described in the literature (Kaiser, C.A., A. Adams, and D.E. Gottschling. 1994. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, N.Y.) using standard procedures.

Result, 2 × 10 ⁵ clones of the mouse embryo library, and to screen 2 × 10 ⁵ clones of the rat brain library, were obtained, respectively 31 and 25 positive clones. Four mouse clones and one rat clone encoded a protein similar to the carboxyl terminal portion of human KIAA0923 (AB023140; GenBank / EMBL / DDBJ). It was found that ADIP specifically bound to the DIL domain of afadin, but did not bind to the DIL domain of yeast Myo4 (FIG. 1B). In addition, the yeast transformant having the illustrated plasmid was streaked on a histidine-deficient synthetic complete medium and incubated at 30 ° C. for 3 days in order to evaluate the HIS3 reporter activity.

  We named this protein ADIP (Afadin DIL domain interacting protein). ADIP has three coiled coil domains, and the region containing the third coiled coil domain (aa339-480) was required for binding of the afadin DIL domain (FIGS. 2A and 3A).

[Example 5] Full length cDNA of mouse ADIP and rat ADIP
BLAST searches were performed against Genbank and EMBL databases for mouse and rat ADIP full-length cDNAs. A selection of hits obtained by BLAST searches against a human subset of Genbank and EMBL sequences was used to assemble the cDNA sequence of the human homologue of KIAA0923 (AB023140; GenBank / EMBL / DDBJ). The cDNA for KIAA0923 is Provided by Dr. Nagase (Kazusa DNA Research Institute) (T. Nagase, K. Ishikawa, M. Suyama, R. Kikuno, M. Hirosawa, N. Miyajima, A. Tanaka, H. Kotani, N. Nomura, and O. Ohara, "Prediction of the coding sequences of unidentified human genes.XIII.The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro.", DNA Res., 6 (1), 63-70 (1999).

  The full-length cDNAs of mADIP (accession number: AF532969) and rADIP (accession number: AF532970) were obtained from mouse and rat brain cDNA (Clontech), respectively, by reverse transcription polymerase chain reaction (PCR) using the following primer sets: For mADIP cDNA, 5′-CGTAGGAGAGTGACAGGAGCTG-3 ′ (SEQ ID NO: 5), 5′-GGTTATCGAGTTTTTCTACATGAC-3 ′ (SEQ ID NO: 6); For rADIP cDNA, 5′-CGTAGGAGAGTGACAGGAGCTG-3 ′ (SEQ ID NO: 5) 7), 5'-TTCCTGTTTTTGCACTGTAGCTG-3 '(SEQ ID NO: 8).

  The PCR product was subcloned into pCR4B (Invitrogen) and nucleotide sequence analysis was performed by the dideoxynucleotide termination method using a DNA sequencer (Model 3100; Applied Biosystems, Inc.).

The mouse and rat full-length cDNAs encoded proteins consisting of 615aa with a calculated molecular weight of 70,954 and 613aa with a calculated molecular weight of 70,684, respectively (FIG. 2A).
The aa sequences of mouse ADIP (mADIP) and rat ADIP (rADIP) were 92% identical to each other and 88% and 87% identical to the human KIAA0923 aa sequence, respectively (FIG. 2A).

Example 6 Transfection To confirm whether the isolated cDNA encodes the full length of ADIP, HEK293 cells were transfected with pCMV-HA-mADIP, which expresses the hemagglutinin (HA) tagged full length mADIP. I did it. For transfection, a CalPhos mammalian transfection kit (Clontech) was used. MDCK and HEK293 cell extracts were subjected to SDS-polyacrylamide gel electrophoresis (PAGE). SDS-PAGE was performed as described in Laemmli (Laemmuli, UK 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 277: 680-685). Subsequently, it was subjected to Western blot using three anti-ADIP pAbs (M57, M01, and M05), which are anti-ADIP polyclonal antibodies (pAbs).

  As a result, a protein having a molecular weight of about 78 kDa similar to the molecular weight of HA-tagged ADIP was detected from the HEK293 cell extract expressing HA-mADIP (FIG. 2B).

  The inventors therefore concluded that the isolated cDNA encodes the full length of ADIP.

[Example 7] ADIP function identification-binding to α-actinin
In order to gain insight into the function of ADIP, we attempted to identify ADIP binding protein (s). As described above, ADIP has three coiled coil domains, and the region containing the third coiled coil domain (aa339-480) of ADIP is necessary for binding to the DIL domain of afadin.

  We performed yeast two-hybrid screening using a region of ADIP as a bait that contains all three coiled coil domains (aa152-436). PGBD-mADIP-B (aa152 to 436), a bait vector, inserts an insert fragment encoding aa residue of mADIP into pGBD-C1 (James, P., J. Hallady, and EA Craig. 1996. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 144: 1425-1436).

We screened 7 × 10 ⁵ clones of the rat lung library and obtained 18 positive clones. Nine clones encoded the C-terminal part of α-actinin-1 (human, BC015766; GenBank). α-actinin is a well-characterized protein that exhibits F-actin cross-linking activity (Burridge, K., and JR Feramisco. 1981. Non-muscle alpha actinins are calcium-sensitive actin-binding proteins. Nature. 294 : 565-567.) Four isoforms of human α-actinin have been identified: non-muscle actinin-1 and actinin-4, and muscle actinin-2 and actinin-3 (Millake, DB, AD Blanchard, B. Patel, and DR Critchley. 1989. The cDNA sequence of a human placental alpha-actinin. Nucleic. Acids. Res. 17: 6725 .; Youssoufian, H., M. McAfee, and DJ Kwiatkowski. 1990. Cloning and chromosomal localization of the human cytoskeletal alpha-actinin Gene reveals linkage to the beta-spectrin gene. Am. J. Hum. Genet 47: 62-71 .; Beggs, AH, TJByers, JH Knoll, FM Boyce, GA Bruns, and LM Kunkel. 1992. Cloning and characterization of two human skeletal muscle alpha-actinin genes located on chromosomes 1 and 11. J. Biol. Chem. 267: 9281-9288 .; Honda, K., T. Yamada, R. Endo, Y. Ino, M. Gotoh, H. Tsuda, Y. Yamada, H. Chiba, and S. Hirohashi. 1998. Actinin-4, a novel actin-bundling protein associated with cell motility and cancer invasion. J. Cell Biol. 140: 1383-1393.).

  As estimated by this analysis, ADIP bound to α-actinin-2 in addition to α-actinin-1 (data not shown). This indicates that the binding of ADIP to α-actinin is not specific for α-actinin-1. In addition, a region containing only the first coiled coil domain (aa1 to 226) of ADIP binds to α-actinin-1 (FIG. 3A), and the two EF hand motifs at the C-terminus of α-actinin-1 represent ADIP It became clear that it was necessary for its binding to (FIG. 9A).

  These results indicate that the first coiled coil domain of ADIP binds to the EF hand motif of α-actinin-1.

Example 8 Immunoprecipitation and affinity chromatography To further confirm the binding of ADIP to afadin in vitro and in vivo, we performed immunoprecipitation and affinity chromatography analysis.

  Co-immunoprecipitation experiments using HEK293 cells were performed as follows; HEK293 cells were transfected with various combinations of expression plasmids. Cells are suspended in 1 ml of buffer A (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10 μM α-phenylmethanesulfonyl fluoride hydrochloride, and 10 μg / ml aprotinin). Sonicate 3 times for 10 seconds at 10 second intervals and incubate on ice for 30 minutes. Centrifugation at 100,000 xg for 15 minutes gave a cell extract (1.2 mg protein) which was then pre-washed by incubation with protein G Sepharose 4 Fast Flow beads (Amersham-Pharmacia Biotechnology). Cell extracts were incubated for 18 hours at 4 ° C. with protein G Sepharose 4 Fast Flow beads coated with 20 μl of anti-FLAG M2 mAb. After washing the beads with buffer A, the beads are boiled for 10 minutes in SDS sample buffer (60 mM Tris-HCl, pH 6.7, 3% SDS, 2% 2-mercaptoethanol, 5% glycerol, pH 6.7). Was eluted. Samples were then subjected to SDS-PAGE followed by Western blot.

  Co-immunoprecipitation experiments using MDCK cells were performed as follows: MDCK cells on two 10 cm dishes were sonicated in 2 ml of buffer A, followed by ultracentrifugation at 100,000 xg for 15 minutes. Went. Cell extracts are prewashed by incubation with protein A Sepharose CL-4B beads (Amersham-Pharmacia Biotechnology) and then with protein A Sepharose CL-4B beads coated with 20 μl of anti-ADIP pAb (M05). Incubated at 4 ° C for 18 hours. After washing the beads with buffer A, the beads were boiled in SDS sample buffer for 10 minutes to elute the bound protein. Samples were then subjected to SDS-PAGE followed by Western blot.

  Affinity chromatography using MDCK cells was performed as follows: MDCK cells on two 10 cm dishes were sonicated in 2 ml of buffer followed by ultracentrifugation at 100,000 xg for 15 minutes. went. The supernatant was incubated for 18 hours at 4 ° C. with MBP or MBP-mADIP (200 pmol each) immobilized on 20 μl (wet volume) amylose resin beads (New England Biolabs). After extensive washing of the beads with buffer A, the bound protein was eluted with buffer A containing 20 mM maltose. The eluate was boiled in SDS sample buffer. Samples were then subjected to SDS-PAGE and then Western blotted with anti-afadin mAb.

  Co-immunoprecipitation of a T7-tagged DIL domain of afadin with FLAG-tagged mADIP-C was performed. The expression vector was transfected into HEK293 cells as illustrated in FIG. 3B. Afadin's T7-tagged DIL domain specifically co-immunoprecipitated with Flag-tagged mADIP-C as shown by Western blot using anti-T7 and anti-Flag mAbs.

  Next, an extract of MDCK cells expressing endogenous afadin was incubated with a full-length mADIP (aa1-615) maltose binding protein (MBP) fusion protein immobilized on amylose-resin beads. After washing the beads with lysis buffer, bound protein was eluted and the eluate was subjected to SDS-PAGE followed by Western blot with anti-Afadin mAb. Afadin actually bound to MBP-mADIP but not to MBP alone (FIG. 3C).

  Finally, to confirm that ADIP binds to afadin in vivo, we examined whether endogenous afadin was co-immunoprecipitated with endogenous ADIP from extracts of MDCK cells. When endogenous ADIP was immunoprecipitated from an extract of MDCK cells using anti-ADIP pAb, endogenous afadin co-immunoprecipitated (FIG. 3D). Afadin did not co-immunoprecipitate with control IgG. Double hybrid analysis and these results showed that ADIP binds to afadin both in vitro and in vivo.

[Example 9] Tissue distribution and subcellular distribution of ADIP Northern blotting was performed as described in the literature (Sambrook, J., EF Fritsch, and T Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 7.1-7.87.). The mADIP cDNA fragment (bp 552-3194) was radiolabeled with [α ³² P] by standard random priming and used as a probe in mouse multiple tissue Northern blots (Clontech).

  As a result, .about.4.3 kb of mRNA was detected in all mouse tissues including heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis (FIG. 4A). A smaller band (˜3.0 kb) was also detected in the liver and testis (FIG. 4A lanes 5 and 8).

  In addition, homogenates of various mouse tissues (each protein 30 μg) were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blot analysis using M57, an anti-ADIP pAb. As a result, a protein of ˜78 kDa was detected. This was the same size as ADIP detected in HEK293 and MDCK cells and mouse spleen, lung and kidney (FIG. 4B lanes 3, 4, 7 and 9). Smaller bands (˜60 kDa in heart, ˜76 kDa and ˜40 kDa in testis) were also detected, suggesting that smaller splice variants of ADIP could be expressed (FIG. 4B lanes 1 and 8).

  Subcellular fraction analysis in rat liver was performed as described in the literature (Kawabe, H., H. Nakanishi, M. Asada, A. Fukuhara, K. Morimoto, M. Takeuchi, and Y. Takai. 2001. Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells. J. Biol. Chem. 276: 48350-48355). Each fraction (each protein 30 μg) was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting using anti-ADIP pAb M57 or anti-afadin mAb. As a result, ADIP was abundant in a rich fraction of AJs and TJs, and aphadin was also abundant here (FIG. 4C lanes 4 and 5). The reason why the protein of ˜78 kDa could not be detected in the liver when Western blot analysis of ADIP tissue distribution was probably due to the low expression level (FIG. 4B lane 5).

  From these results, it was shown that the expression level of ADIP varies widely depending on the tissue but is widely expressed.

Example 10 Co-localization of afadin and ADIP in AJs of epithelial cells Afadin has been shown to be strictly localized to AJs lined with F-actin bundles (Mandai et al., J. Cell Biol. 1997, 139: 517-528). Therefore, the present inventors examined by immunofluorescence microscopy whether ADIP was colocalized with afadin in AJs of MDCK cells.

  Immunofluorescence microscopy of cultured cells and frozen sections of mouse tissues was performed as described in the literature (Mandai, K., H. Nakanishi, A. Satoh, H. Obaishi, M. Wada, H. Nishioka, M. Itoh, A. Mizoguchi, T. Aoki, T. Fujimoto, Y. Matsuda, S. Tsukita, and Y. Takai. 1997. Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to -cell adherens junction. J. Cell Biol. 139: 517-528 .; Takahashi, K., H. Nakanishi, M. Miyahara, K. Mandai, K. Satoh, A. Satoh, H. Nishioka, J. Aoki, A. Nomoto, A. Mizoguchi, and Y. Takai. 1999. Nectin / PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein. J. Cell Biol. 145: 539-549.). Specifically, MDCK cells were double-stained with various combinations of anti-ADIP (MO5), anti-afadin, and anti-vinclin Abs.

  As a result, ADIP and afadin colocalized at the cell-cell junction (FIG. 5A). Essentially similar results were obtained with the three anti-ADIP pAbs, M57, M01, and M05.

  Furthermore, ADIP was co-stained with the Golgi 58k protein, which is a marker for the Golgi complex. The result was stained in the Golgi complex in the perinuclear region (FIG. 5A).

[Example 11] Comparison of localization of ADIP and endogenous afadin
In order to confirm the co-localization of ADIP and afadin, nectin-2α-L and nectin-2α-ΔC-L cells stably expressing HA-tagged ADIP (rADIP-C; aa159-613) were expressed in LIPOFECTAMINE Prepared using 2000 (Invitrogen).

  One of cadherin-deficient L cells stably expressing full-length Nectin-2α (Nectin-2α-L cells) or Nectin-2α (Nectin-2α-ΔC-L cells) lacking four C-terminal aa Overexpressed and expressed protein localization compared to endogenous afadin localization. Full-length Nectin-2α can bind to afadin, but Nectin-2α-ΔC cannot bind (Takahashi, K., H. Nakanishi, M. Miyahara, K. Mandai, K. Satoh, A. Satoh, H. Nishioka, J. Aoki, A. Nomoto, A. Mizoguchi, and Y. Takai. 1999. Nectin / PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein. Cell Biol. 145: 539-549.).

  As a result, as described in the literature, afadin was found to be as described in the literature (Miyahara, M., H. Nakanishi, K. Takahashi, K. Satoh-Horikawa, K. Tachibana, and Y. Takai. 2000. clustering at cell-cell contact sites but not for its cis dimerization or trans interaction. J. Biol. Chem. 275: 613-618 .; Tachibana, K., H. Nakanishi, K. Mandai, K. Ozaki, W. Ikeda , Y. Yamamoto, A. Nagafuchi, S. Tsukita, and Y. Takai. 2000. Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins. J. Cell Biol. 150: 1161-1176.) In Nectin-2α-L cells, they concentrated on cell-cell adhesion sites based on Nectin-2α. Endogenously expressed HA-ADIP was concentrated at the cell-cell adhesion site based on Nectin-2α, and co-localized with afadin in Nectin-2α-L cells (FIG. 6A).

  However, endogenously expressed HA-ADIP was not concentrated at the cell-cell adhesion site in Nectin-2α-ΔC-L cells (FIG. 6B).

Example 12 Accurate Localization of ADIP in the Junction Complex Region To examine the exact localization of ADIP in the junction complex region, frozen sections of mouse small intestine were analyzed for anti-ADIP pAb, anti-afadin mAb, and anti-IPA. Triple staining with ZO-1 mAb. The small intestine frozen sections were used because TJs and AJs were well separated in this cell type (Itoh, M., A. Nagafuchi, S. Yonemura, T Kitani-Yasuda, S. Tsukita, and S. Tsukita. 1993. The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy. J Cell Biol. 121: 491-502. ZO-1 is known to be a marker of TJs (Stevenson, BR, JD Siliciano, MS Mooseker, and DA Goodenough. 1986. dentification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J. Cell Biol. 103: 755-766 .; Itoh, M., A. Nagafuchi, S. Yonemura, T Kitani-Yasuda, S. Tsukita, and S. Tsukita. 1993. The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy. J Cell Biol. 121: 491-502.

  Immunoelectron microscopy of mouse intestinal absorptive epithelial cells using ultrathin cryosectioning was performed as described in the literature (Mandai, K., H. Nakanishi, A. Satoh, H. Obaishi, M. Wada, H Nishioka, M. Itoh, A. Mizoguchi, T. Aoki, T. Fujimoto, Y. Matsuda, S. Tsukita, and Y. Takai. 1997. Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin -based cell-to-cell adherens junction. J. Cell Biol. 139: 517-528.).

  As a result, ADIP showed co-localization with afadin, but in the absorptive epithelium, it was localized slightly to the basal side than ZO-1 (arrows in FIGS. 7A and 7B).

  In addition, it was found that ADIP was exclusively localized in AJs lined with F-actin bundles and was absent in TJs and desmosomes (FIG. 7C). This localization pattern of ADIP was the same as that of afadin and nectin. However, it was concentrated in AJs, but it was different from the localization pattern of E-cadherin, which is more widely distributed from the tip of the lateral plasma membrane to the basal side (Mandai, K., H. Nakanishi, A Satoh, H. Obaishi, M. Wada, H. Nishioka, M. Itoh, A. Mizoguchi, T. Aoki, T. Fujimoto, Y. Matsuda, S. Tsukita, and Y. Takai. 1997. Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction. J. Cell Biol. 139: 517-528 .; Takahashi, K., H. Nakanishi, M. Miyahara, K. Mandai , K. Satoh, A. Satoh, H. Nishioka, J. Aoki, A. Nomoto, A. Mizoguchi, and Y. Takai. 1999. Nectin / PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein. J. Cell Biol. 145: 539-549.).

  These results indicate that ADIP co-localizes with afadin and nectin in cell-cell AJs lined with F-actin bundles.

[Example 13] ADIP signal
The ADIP signal was not detected at the focal adhesion site where a vinculin signal was detected in MDCK cells (FIG. 5B).

  In the heart, a localized adhesion site, called the Costamea, was well-developed and located periodically along the lateral border of cardiomyocytes (Terracio, L., DG Simpson, L. Hilenski, W. Carver, RS Decker, N. Vinson, and TK Borg. 1990. Distribution of vinculin in the Z-disk of striated muscle: analysis by laser scanning confocal microscopy. J. Cell Physiol. 145: 78-87.). It is known that vinculin is localized in Costa mer, while nectin and afadin are not (Mandai, K., H. Nakanishi, A. Satoh, H. Obaishi, M. Wada, H. Nishioka, M. Itoh, A. Mizoguchi, T. Aoki, T. Fujimoto, Y. Matsuda, S. Tsukita, and Y. Takai. 1997. Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to -cell adherens junction. J. Cell Biol. 139: 517-528 .; Takahashi, K., H. Nakanishi, M. Miyahara, K. Mandai, K. Satoh, A. Satoh, H. Nishioka, J. Aoki, A. Nomoto, A. Mizoguchi, and Y. Takai. 1999. Nectin / PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein. J. Cell Biol. 145: 539-549.).

  The ADIP signal was not detected in the Costa mer where the vinculin signal was detected (arrow in FIG. 7D).

  Both ADIP and vinculin signals were detected on an intercalated disc corresponding to cell-cell AJs (wedge in FIG. 7D).

  These results indicated that ADIP does not localize at the cell-matrix junction.

Example 14 Ca ²⁺ Switch Assay Next, we investigated the localization of ADIP compared to the localization of afadin during the destruction and formation of AJs in MDCK cells.

Ca ²⁺ switch experiments using MDCK cells were performed as described in the literature (Kartenbeck, J., M. Schmelz, WW Franke, and B. Geiger. 1991. Endocytosis of junctional cadherins in bovine kidney epithelial (MDBK) cells cultured in low Ca ²⁺ ion medium. J. Cell Biol. 113: 881-892.). Briefly, MDCK cells (1 × 10 ⁵ ) were seeded on 18 mm glass coverslips in 12 well culture dishes. After 48 hours, the cells were washed with phosphate buffered saline (PBS) and cultured in 2 mM Ca ^{2+ for} 60 minutes in DMEM without serum. Next, the cells were cultured in 2 μM Ca ²⁺ (DMEM containing 5 mM EGTA) for 120 minutes. After culturing, the cells were washed with PBS and cultured again in 2 mM Ca ^{2+ in} DMEM without serum for 60 minutes. If cells were treated with phorbol ester, ie 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the cells were washed with PBS and 60 minutes in 2 mM Ca ^{2+ in} DMEM without serum Cultured. The cells were then incubated for 120 minutes with 2 μM Ca ²⁺ containing 5 mM EGTA. After incubation, 100 nM TPA was added to the medium and the cells were further cultured for 60 minutes.

As a result, when the Ca ²⁺ concentration in the culture medium was switched from 2 mM to 2 μM, the MDCK cells were obtained as described in the literature (Kartenbeck, J., M. Schmelz, WW Franke, and B. Geiger. 1991. Endocytosis of junctional cadherins in bovine kidney epithelial (MDBK) cells cultured in low Ca ²⁺ ion medium. J. Cell Biol. 113: 881-892.). Afadin, E-cadherin and ADIP signals were highly concentrated at the cell-cell adhesion site of cells cultured in 2 mM Ca ²⁺ (FIG. 8A). When these cells were cultured in 2 μM Ca ²⁺ for 120 minutes, the E-cadherin signal disappeared and partly appeared on intracellular vesicles (E-cadherin in FIG. 8B). Most of the afadin signals remain on the free surface of the plasma membrane, as described in the literature (Sakisaka, T, H. Nakanishi, K. Takahashi, K. Mandai, M. Miyahara, A. Satoh, K. Takaishi, and Y. Takai. 1999. Different behavior of l-afadin and neurabin-II during the formation and destruction of cell-cell adherens junction. Oncogene. 18: 1609-1617 .; Fukuhara, A., K. lrie, H. Nakanishi, K. Takekuni, T Kawakatsu, W. Ikeda, A. Yamada, T. Katata, T. Honda, T. Sato, K. Shimizu, H. Ozaki, H. Horiuchi, T. Kita, and Y. Takai. 2002. Involvement of Nectin in the Localization of Junctional Adhesion Molecule at Tight Junctions Oncogene. In press.), A ring-like structure was formed (Afadin in FIG. 8B). Under these conditions, most of the ADIP signal disappeared from the free surface of the plasma membrane (ADIP in FIG. 8B).

Next, MDCK cells pre-cultured for 120 minutes with 2 μM Ca ²⁺ were re-cultured for 60 minutes with 2 mM Ca ²⁺ to examine ADIP accumulation at the cell-cell adhesion site compared to E-cadherin accumulation. It was. After incubation with 2 mM Ca ²⁺ , the E-cadherin signal refocused on the cell-cell adhesion site where afadin is localized (E-cadherin in FIG. 8C). The ADIP signal was also concentrated again at the cell-cell adhesion site (ADIP in FIG. 8C).

Next, when we incubate MDCK cells pre-cultured with 2 μM Ca ²⁺ for 120 minutes with 2 μM Ca ²⁺ for 60 minutes with TPA, a TJ-like structure was formed, and afadin and ZO-1 It was shown that E-cadherin does not accumulate (Balda, MS, L. Gonzalez-Mariscal, K. Matter, M. Cereijido, and JM Anderson. 1993. Assembly of the tight junction: the role of diacylglycerol. J. Cell Biol. 123: 293-302 .; Asakura, T, H. Nakanishi, T Sakisaka, K. Takahashi, K. Mandai, M. Nishimura, T. Sasaki, and Y. Takai. 1999. Similar and differential behavior between the nectin-afadin-ponsin and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells. Genes Cells. 4: 573-581 .; Fukuhara, A., K. lrie, H. Nakanishi , K. Takekuni, T Kawakatsu, W. Ikeda, A. Yamada, T. Katata, T. Honda, T. Sato, K. Shimizu, H. Ozaki, H. Horiuchi, T. Kita, and Y. Takai. 2002 Involvement of Nectin in the Localization of Junctional Adhesion Molecule at Tight Junctions Oncogene. In press .; Fukuhara, A., K. Irie, A. Yamada, T Katata, T. Honda, K. Shimizu, H. Nakanishi, and Y. Takai. 2002 Role of Nectin in Organization of Tight Junctions in Epithelial Cells. Genes Cells. In press. In addition, the afadin signal was localized in the TPA-induced TJ-like structure, but the ADIP or E-cadherin signal was not localized (FIG. 8D).

  These results indicate that ADIP exhibits a different localization behavior from that of afadin during fracture and formation of the junction complex.

Example 15 In vitro and in vivo binding of ADIP to α-actinin-1
In order to confirm the binding of ADIP to α-actinin-1 in vitro and in vivo, we performed immunoprecipitation analysis.

  HEK293 cells were treated with α-actinin-1 HA-tagged C-terminal portion (HA-α-actinin-1-C; aa406-892) and FLAG-tagged ADIP (FLAG-mADIP-M; aa152-436). Simultaneously transfected.

  When FLAG-tagged ADIP was immunoprecipitated from cell extracts using anti-FLAG mAb, HA-α-actinin-1-C was co-immunoprecipitated as detected by Western blot using HA mAb ( FIG. 9B).

  When FLAG-mADIP-M was immunoprecipitated from an extract of HEK293 cells that transiently expressed only FLAG-tagged mADIP (FLAG-mADIP-M; aa152-436) using anti-FLAG Ab, Endogenous α-actinin co-immunoprecipitated (FIG. 9C).

  Finally, when endogenous ADIP was immunoprecipitated from an extract of MDCK cells using anti-ADIP pAb, endogenous α-actinin co-immunoprecipitated (FIG. 9D). α-actinin did not co-immunoprecipitate with control IgG.

  These results indicated that ADIP binds to α-actinin both in vitro and in vivo.

[Example 16] Co-localization of α-actinin and ADIP in AJs of MDCK cells Since α-actinin interacts with α-catenin and is localized to AJs (Knudsen, KA, AP Soler, KR Johnson, and MJ Wheelock. 1995. Interaction of alpha-actinin with the cadherin / catenin cell-cell adhesion complex via alpha-catenin. J. Cell Biol. 130: 67-77.) We examined whether ADIP co-localized with α-actinin in AJs of MDCK cells by immunofluorescence microscopy.

  As a result, ADIP and α-actinin co-localized at the cell-cell junction (FIG. 10A). α-actinin was also localized to the localized adhesion site, whereas ADIP was not (data not shown).

  To further confirm the co-localization of ADIP and α-actinin in the junction complex region, frozen sections of mouse small intestine were double stained with anti-ADIP pAb and anti-α-actinin mAb.

  As a result, ADIP co-localized with α-actinin in the absorptive epithelium (FIG. 10B). The localization of ADIP was strictly limited to AJs as described above, while the localization of α-actinin was more widely distributed from the tip of the lateral plasma membrane to the basal side (FIG. 10B).

  These results indicate that ADIP forms a complex with afadin and α-actinin in cell-cell AJs lined with F-actin bundles.

FIG. 2 is a diagram and a photograph showing the binding of ADIP to the DIL domain of afadin. (A): Schematic structure of afadin. RA, Ras association domain; FHA, forkhead association domain; DIL, dilution domain; PDZ, PDZ domain; PR, proline-rich domain. (B): Yeast double hybrid assay showing specific binding of ADIP to the DIL domain of afadin. It is the figure and photograph which show the amino acid sequence of ADIP. (A): deduced amino acid sequences of rat ADIP, mouse ADIP and human KIAA0923. Gray fills represent the same sequence. The putative coiled coil domain is underlined. (B): Comparison of molecular weights of natural and recombinant ADIP proteins. pCMV HA mADIP was transfected into HEK293 cells and cell lysates were run on SDS-PAGE (10% polyacrylamide gel) followed by Western blot using anti-ADIP Ab (M57). Control lysates of HEK293 cells and MDCK cells were similarly subjected to SDS-PAGE, followed by Western blotting. Lane 1, control HEK293 cells (protein 1 μg); lane 2, pCMV HA mADIP-transfected HEK293 cells (protein 1 μg); and lane 3, MDCK cells (protein 1 μg). FIG. 2 is a diagram and photographs showing in vitro and in vivo binding of ADIP to afadin. (A): Schematic structure of mouse ADIP. CC, coiled coil domain. Yeast double hybrid analysis of afadin or α-actin binding region of ADIP. ADIP, afadin (DIL domain) and α-actinin were constructed in pGBDU or pGAD and cotransformed into reporter yeast strains. Binding of ADIP to afadin or α-actinin was measured by assessing growth on synthetic complete medium lacking histidine and adenine, as shown by expression of HIS3 and ADE2 reporter genes. +, With interaction;-, without interaction; and NT, not tested. (B): Co-immunoprecipitation of T7-tagged DIL domain of afadin with FLAG-tagged mADIP-C. The expression vector was transfected into HEK293 cells as shown. Afadin's T7-tagged DIL domain specifically co-immunoprecipitated with Flag-tagged mADIP-C as shown by Western blot using anti-T7 and anti-Flag mAbs. (C): In vitro binding of afadin to MBP-mADIP. MDCK cell extracts were incubated with MBP or MBP-mADIP (full length) immobilized on amylose resin beads. The beads were then subjected to SDS-PAGE (10% polyacrylamide gel) followed by Western blot using anti-afadin mAb. (D): Co-immunoprecipitation of endogenous afadin with endogenous ADIP by MDCK cells. MDCK cell lysates were immunoprecipitated with anti-ADIP antibody (M05) and analyzed by Western blot using anti-ADIP pAb (M05) and anti-Afadin mAB. Results are representative of 3 independent experiments. It is a photograph which shows the structure | tissue and subcell distribution of ADIP. (A): Northern blot analysis. Mouse RNA blot membrane (Clontech) was hybridized with a ³² P-labeled fragment of ADIP cDNA (bp 552-3194) followed by autoradiography. Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; and lane 8, testis (short-term exposure). (B) Western blot analysis. Various mouse tissue homogenates (each protein 30 μg) were run on SDS-PAGE (10% polyacrylamide gel) followed by Western blot using anti-ADIP Ab (M57). Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis and lane 9, MDCK cells. (C): ADIP subcellular distribution in rat liver. Sub-cell fractionation of rat liver is performed, each fraction (30 μg protein each) is run on SDS-PAGE (10% polyacrylamide gel), followed by Western blot using anti-ADIP pAb (M57) or anti-afadin mAb went. Lane 1, homogenate fraction; lane 2, soluble fraction; lane 3, pellet fraction; lane 4, fraction rich in capillary bile ducts; and lane 5, fraction rich in AJs and TJs. Results are representative of 3 independent experiments. It is a photograph which shows the localization of ADIP in AJs in MDCK cell. MDCK cells were double-stained with various combinations of anti-ADIP (M05), anti-afadin, and anti-vinclin Abs. (A) ADIP and afadin; (B) ADIP and vinculin. The bar is 10 μm. Results are representative of 3 independent experiments. It is a photograph showing the co-localization of ADIP with afadin in Nectin-2α-L cells. (A): ADIP supplementation of cell-cell adhesion sites based on Nectin-2α. Nectin-2α-L cells that transiently express HA-tagged ADIP (rADIP-C; aa159-613) were double-stained with anti-HA and anti-afadin mAbs. Arrows are cell-cell adhesion sites based on Nectin-2α. (B): No replenishment of ADIP to cell-cell adhesion sites based on Nectin-2α-ΔC. Nectin-2α-ΔC-L cells that transiently express HA-tagged ADIP (rADIP-C; aa159-613) were double-stained with anti-HA and anti-afadin mAb. Arrows indicate cell-cell adhesion sites based on Nectin-2α-ΔC. The bar is 10 μm. Results are representative of 3 independent experiments. It is a photograph which shows the co-localization of ADIP and afadin in AJs in mouse | mouth small intestine absorptive epithelial cells. (A) and (B): localization of ADIP, afadin and ZO-1. In (A), frozen sections were triple stained with anti-ADIP pAb (M01) and anti-afadin and anti-ZO-1 mAb. In (B), frozen sections were double-stained with anti-ADIP pAb (M01) and anti-afadin mAb (left) and with anti-ADIP pAb (M01) and anti-ZO-1 mAb (right). Arrow indicates ADIP signal colocalized with afadin signal. The arrow is an ADIP signal localized slightly on the basal side of the ZO-1 signal. The bar is 10 μm. (C): Ultrafine localization of ADIP in mouse intestinal absorptive epithelial cells. Mouse intestinal absorptive epithelial cells were labeled with anti-ADIP pAb (M01) using ultrathin cryosectioning techniques. AJ, adhesive bond; DS, desmosome; TJ, tight junction; and bars 0.1 μm. (D): Absence of ADIP in cell matrix binding in mouse heart cells. Cryosections were double-stained with anti-ADIP pAb (M01) and anti-vinclin mAb. Arrows are intervening plates; arrows are Costamere; and bars are 50 μm. Results are representative of 3 independent experiments. It is a photograph which shows the behavior of ADIP during destruction and formation of the junction complex which consists of AJs and TJs in MDCK cell. MDCK cells were tristained with anti-ADIP (M05), anti-afadin and anti-E-cadherin Ab. (A): localization of ADIP, afadin and E-cadherin in cells cultured in 2 mM Ca ²⁺ (normal Ca ²⁺ ). (B): Disappearance of the ADIP immunofluorescence signal from the plasma membrane due to a decrease from normal to low concentration of media Ca ²⁺ . MDCK cells were cultured for 120 minutes in 2 μM Ca ²⁺ (low Ca ²⁺ ). (C): ADIP recruitment to the cell-cell adhesion site by increasing the Ca ²⁺ concentration from low to normal. MDCK cells were incubated with 2 μM Ca ^{2+ for} 120 minutes and then incubated with 2 mM Ca ²⁺ for 60 minutes (low Ca ²⁺ + normal Ca ²⁺ ). (D): No ADIP supplementation to TPA-induced TJ-like structure. MDCK cells were incubated with 2 μM Ca ^{2+ for} 120 minutes and then incubated with 100 nM TPA for 60 minutes (low Ca ²⁺ TPA). The bar is 10 μm. Results are representative of 3 independent experiments. FIG. 6 is a photograph showing in vivo binding of ADIP to α-actinin. (A): Schematic structure of α-actinin-1. CH, calponin homolog domain; SPEC, spectrin-like repeats; and EF, EF hand. Yeast double hybrid analysis of the ADIP binding domain of α-actinin-1. Binding of α-actinin-1 to ADIP was measured by assessing growth on synthetic complete medium lacking histidine and adenine, respectively, as shown by expression of HIS3 and ADE2 reporter genes. +, With interaction; +/-, with weak interaction; and-, without interaction. (B): Co-immunoprecipitation of HA-tagged α-actinin-1-C and Flag-tagged mADIP-M. The expression vector was transfected into HEK293 cells as shown. HA-tagged α-actinin-1-C specifically co-immunoprecipitated with Flag-tagged mADIP-M as shown by Western blot with anti-HA and anti-Flag mAb. (C): Co-immunoprecipitation of endogenous α-actinin and Flag-tagged mADIP-M. The expression vector was transfected into HEK293 cells as shown. Endogenous α-actinin-1 specifically co-immunoprecipitated with Flag-tagged mADIP-M as shown by Western blot with anti-α-actinin pAb and anti-Flag mAb. (D): Co-immunoprecipitation of endogenous α-actinin and endogenous afadin with endogenous ADIP by MDCK cells. MDCK cell lysates were immunoprecipitated with anti-ADIP antibody (M05) and analyzed by Western blot using anti-ADIP (M05), anti-α-actinin and anti-Afadin Ab. Results are representative of 3 independent experiments. It is a photograph showing the localization of α-actinin and ADIP in AJs in MDCK cells and mouse small intestinal absorptive epithelial cells. (A): Localization of ADIP and α-actinin in MDCK cells. Cells were double stained with anti-ADIP pAB (M05) and anti-α-actin mAb. (B): Localization of ADIP and α-actinin in mouse small intestinal absorptive epithelial cells. Cryosections were triple stained with anti-ADIP pAb (M01) and anti-α-actin mAb. The bar is 10 μm. Results are representative of 3 independent experiments.

Claims (7)

A polynucleotide encoding a protein consisting of the amino acid sequence at positions 1 to 226 of the amino acid sequence set forth in SEQ ID NO: 2 or 4.   A polypeptide encoded by the polynucleotide of claim 1.   A vector into which the polynucleotide according to claim 1 is inserted.   A host cell carrying the polynucleotide of claim 1 or the vector of claim 3.   The method for producing a polypeptide according to claim 2, comprising culturing the host cell according to claim 4 and recovering the produced polypeptide from the host cell or a culture supernatant thereof. An actinin binding agent comprising a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4. A method for screening a candidate compound for an agent that regulates an actin skeleton, comprising the following steps (a) to (c):
(a) contacting actinin with a polypeptide encoded by a polynucleotide according to any one of the following (i) to (iii) and a test compound;
(i) a polynucleotide encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4
(ii) a polynucleotide comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 3
(iii) a polynucleotide comprising an amino acid sequence having 95% or more identity with the amino acid sequence of SEQ ID NO: 2 or 4, and comprising a base sequence encoding a protein having a binding activity to actinin
(b) measuring the actinin, the binding activity between the polypeptide,
(c) a step of selecting a compound that changes the binding activity as compared with the case where the test compound is not contacted

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