SIGNAL TRANSDUCTION BY INTEGRIN-MATRIX INTERACTION

整合素-基质相互作用的信号转导

• 批准号: 10044338
• 负责人: SEKIGUCHI Kiyotoshi
• 金额: $ 2.94万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044338/
• 关键词: BASEMENT MEMBRANE; LAMININ; INTEGRIN; CELL ADHESION; CYTOSKELETON; TRANSMEMBRANE 4 SUPERFAMILY; シグナル伝達

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 … More 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

研究了细胞-基质相互作用的分子机制，特别是整合素介导的细胞外基质信号转导，特别强调了interin α3β1与基底膜蛋白层粘连蛋白（LN）的特异性相互作用。主要研究结果如下。(1)LN-8的纯化和鉴定：我们通过RT-PCR在超过35个人类细胞系中筛选了五种不同LN α链的表达。T98 G胶质瘤细胞仅表达5条LN α链中的α4链。我们大规模培养T98 G细胞，收获用过的培养基（约4升），通过硫酸铵沉淀浓缩，然后进行凝胶过滤和使用抗LN β1单克隆抗体4F 5的免疫亲和层析。在非还原条件下，所得蛋白在SDS-PAGE上明显是均一的，并且由于其由α4/β1/γ 1链组成，因此被鉴定为LN-8。纯化的LN-8在介导粘附方面的作用较弱， ...更多信息 LN-5和LN-10/11对T98 G细胞的粘附活性明显高于LN-5和LN-10/11，与LN-1的粘附活性相当。细胞粘附到LN-8上是通过整合素α6β1和α3β1介导的，这两种整合素是两种主要的LN结合整合素。(2)LN-10/11的细胞骨架调节：我们检查了粘附到LN-10/11上是否可以诱导应力纤维和局灶性粘连的形成。尽管其非常有效的细胞粘附活性，LN-10/11未能诱导应力纤维或局灶性粘附在LN-10/11粘附的细胞。我们还发现，在LN-10/11上未检测到rho的激活，这与其诱导应力纤维和局灶性粘连的失败一致。(3)与整合素α3β1紧密结合的30 kDa蛋白质作为CD 151的鉴定：我们制备了针对整合素α3亚基胞质结构域的多克隆抗体，并从人胎盘中纯化了整合素α3β1。纯化的整合素α3β1在SDS-PAGE上有三条主要带，即：e.对应于α3和β1链的150 kDa/120 kDa条带和非预期的30 kDa条带。通过使用由Hitoshi Hasegawa博士（Ehime University Medical School）提供的抗CD 151抗体的免疫沉淀，将30 kDa条带鉴定为CD 151，其为跨膜4超家族蛋白之一。我们还制备了两种均识别30 kDa蛋白质的单克隆抗体，并且基于它们与用CD 151 cDNA转染的NIH 3 T3细胞的反应性，证实了所述抗体识别CD 151。这些单克隆抗体应被证明是有用的研究的生理功能的CD 151。(4)LN α3链基因敲除小鼠的产生：我们成功地产生了缺乏LN α3链基因的小鼠.这些小鼠在子宫内死亡，并伴有严重的上皮异常。突变小鼠的基底膜不能通过整合素α6β4诱导稳定的粘附，这与存在连接性水泡和异常的半桥粒一致。我们还在表皮基底膜中检测到整合素α3β1的新配体，其在LN-5不存在时起作用。我们还在表皮基底膜中检测到整合素α3β1的新配体，该配体在LN-5缺乏的情况下起作用。我们还鉴定了突变上皮细胞的存活缺陷，该缺陷可以被外源性LN-5、胶原或抗α6β4的抗体拯救，这表明通过α6或β4整合素的信号传导足以维持存活。少

项目成果

Miyado, K. et al.: "Requirement of CD9 on the egg plasma mambrane for fertilization"Nature. 287. 321-324 (2000)

Miyado, K. 等人：“受精时卵浆膜上 CD9 的要求”《自然》。

Miyado, K. et al.: "Requirement of CD9 on the egg plasma membrane for fertilization"Nature. 287. 321-324 (2000)

Miyado, K. 等人：“受精时卵质膜上 CD9 的要求”《自然》。

Koshikawa, N., Giannelli, G., Cirulli, V., Miyazaki, K., and Quaranta, V.: "Role of cell surface metalloproteinase MT1-MMP in epithelial cell migration over laminin-5."J. Cell Biol.. 148. 615-624 (2000)

Koshikawa, N.、Giannelli, G.、Cirulli, V.、Miyazaki, K. 和 Quaranta, V.：“细胞表面金属蛋白酶 MT1-MMP 在层粘连蛋白 5 上皮细胞迁移中的作用。”

Lzumi, Y., Hirata, M., Hasuwa, H., Iwamoto, R., Umata, T., Miyado, K., Tamai, Y., Kurisaki, T., Sehara-Fujisawa, A., Ohno, S. and Mekada, E.: "A metalloprotease-disintegrin, MDC9/Meltrin-γ/ADAM9, and PKCδare involved in TPA-induced ectodomain shedding of

Lzumi, Y.、Hirata, M.、Hasuwa, H.、Iwamoto, R.、Umata, T.、Miyado, K.、Tamai, Y.、Kurisaki, T.、Sehara-Fujisawa, A.、Ohno, S和 Mekada, E.：“金属蛋白酶解整合素、MDC9/Meltrin-γ/ADAM9 和 PKCδ 参与 TPA 诱导的胞外域脱落。

Kikkawa, Y. et al.: "Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by α3β1,α6β1,and ,α6β4 integrins"J. Cell Sci.. 113. 869-876 (2000)

Kikkawa, Y. 等人：“层粘连蛋白 10/11 的整合素结合特异性：层粘连蛋白 10/11 被 α3β1、α6β1 和 α6β4 整合素识别”J. Cell Sci.. 113. 869-876 (2000)