| Project/Area Number |
10044338
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Osaka University |
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Principal Investigator |
SEKIGUCHI Kiyotoshi Inst. Protein Res., Osaka University Professor, 蛋白質研究所, 教授 (50187845)
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| Co-Investigator(Kenkyū-buntansha) |
MEKADA Eisuke Inst. Life Sci., Kurume Univ. Professor, 分子生命科学研究所, 教授 (20135742)
TUJI Tsutomu Hoshi University Professor, 教授 (00143503)
MIYAZAKI Kaoru Yokohama City University Professor, 木原生物学研究所, 教授 (70112068)
GU Jianguo Inst. Protein Res., Osaka Univ. Instructor, 蛋白質研究所, 助手 (30314420)
眞鍋 理一郎 大阪府立母子保健総合医療センター研究所, 研究員 (30280837)
CARTER Willi フレッドハッケンソン癌研究所(米国), 部長
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1998: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | BASEMENT MEMBRANE / LAMININ / INTEGRIN / CELL ADHESION / CYTOSKELETON / TRANSMEMBRANE 4 SUPERFAMILY / シグナル伝達 |
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| Research Abstract |
Molecular mechanisms of cell-matrix interaction, particularly integrin-mediated signal transduction from extracellular matrices, were investigated with special emphasis on the specific interaction of interin α3β1 with the basement membrane protein laminin (LN). Major findings obtained are as follows.
(1) Purification and Characterization of LN-8 : We screened the expression of five different LN αchains in more than 35 human cell lines by RT-PCR. The T98G glioma cells were found to express only α4 chain among the five LN α chains. We grew T98G cells in a large scale and harvested the spent medium (approximately 4 liter) which was concentrated by ammonium sulfate precipitation, followed by gel filtration and immunoaffinity chromatography using the anti-LN β1 monoclonal antibody 4F5. The resulting protein was apparently homogeneous on SDS-PAGE under nonreducing conditions and identified to be LN-8 since it consisted of α4/β1/γi chains. The purified LN-8 was less potent in mediating adhesio
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n of T98G cells than LN-5 and LN-10/11 and comparable to LN-1 in its cell-adhesive activity. Cell adhesion onto LN-8 was mediated through integrin α6β1 and α3β1, two major LN-binding integrins.
(2) Cytoskeletal Modulation on LN-10/11 : We examined whether adhesion onto LN-10/11 could induce formation of stress fibers and focal adhesions. Despite its very potent cell-adhesive activity, LN-10/11 failed to induce stress fibers or focal adhesions in cells adhered to LN-10/11. We also found that activation of rho was not detected on LN-10/11, consistent with its failure in inducing stress fibers and focal adhesions.
(3) Identification of the 30 kDa Protein That Tightly Associates with Integrin α3β1 As CD151 : We produced polyclonal antibodies against the cytoplasmic domain of the integrin α3 subunit and purified integrin α3β1 from human placenta. Integrin α3β1 thus purified gave three major bands on SDS-PAGE, i. e. 150 kda/120 kDa bands corresponding to the α3 and β1 chains and an unexpected 30 kDa band. The 30 kDa band was identified as CD151, one of the transmembrane 4 superfamily proteins, by immunoprecipitation using the anti-CD151 antibody provided by Dr. Hitoshi Hasegawa (Ehime University Medical School). We also produced two monoclonal antibodies both recognizing the 30 kDa protein and the antibodies were confirmed to recognize CD151 based on their reactivity with the NIH3T3 cells transfected with the CD151 cDNA. These monoclonal antibodies should prove to be useful in the study of physiological functions of CD151.
(4) Production of LN α3 Chain Knockout MIce : We succeeded in producing mice lacking the LN α3 chain gene. The mice died neonatally with profound epithelial abnormalities. The basement membrane of the mutant mice could not induce stable adheison by integrin α6β4, consistent with the presence of junctional blisters and abnornmal hemidesmosomes. We also detected a new ligand for integrin α3β1 in the epidermal basement membrane that worked in the absence of LN-5. We also detected a new ligand for integrin α3β1 in the epidermal basement membrane that worked in the absence of LN-5.We also identified a survival defect in mutant epithelial cells that could be rescued by exogenous LN-5, collagen, or antibody against α6β4, suggesting that signaling through α6 or β4 integrins is sufficient for survival. Less
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