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Studies on the Regulatory Mechanisms and Molecular Diversity of Integrinmediated Signal Transduction

整合素介导的信号转导调控机制及分子多样性研究

基本信息

批准号：
12480189
负责人：
SEKIGUCHI Kiyotoshi
金额：
$ 7.74万
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：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Molecular mechanisms underlying the cell-basement membrane interaction were investigated with an emphasis on the integrin-mediated signal transduction. The major achievements are summarized below.1. The major integrin isoforms responsible for cell adhesion onto basement membranes, i.e., integrin α3β1 and α6β1, were purified to homogeneity under non-denaturing conditions. The ligand binding specificities of these integrins were determined by solid-phase binding assays towards a panel of laminin isoforms differing in their α subunit composition. Our results clearly showed that laminin-10 that contains the α5 chain is the most preferred ligand for both laminin-binding integrins.2. Cell adhesion onto laminin-10 through these integrins was found to selectively activate Rac, a member of Rho family GTPases, thereby strongly promoting formation of lamellipodia and cell motility. Rac activation on laminin-10 was associated with Selective tyrosine-phosphorylation of p130Cas, but not FAK, followe … More d by a ternary complex formation among p130Cas, CrkII, and DOCK180, the latter of which is a guanine nucleotide exchange factor for Rac.3. Cell adhesion onto laminin-10 was also found to activate the PI3K-Akt pathway, leading to a strong activation of Akt, Consistent with the role of Akt in cell survival, cells on laminin-10 were much more resistant than those on fibronectin against apoptosis induced. By, serum deprivation. Interestingly, cell survival on fibronectin was dominantly dependent on the ERK pathway, but not the PI3K-Akt pathway.4. Laminin-8, the major laminin isoform in the endothelial basement membrane, was purified to homogeneity from the conditioned medium of human glioma cells. Cell adhesion onto laminin-8 was strictly dependent on integrin α6β1 and preferentially activated Rac, as was the case with cell adhesion onto laminin-10.5. A 30 kD protein was found to copurify with integrin α3β1. This 30 kD protein was identified as CD151, a member of tetraspanin family proteins that span the plasma membrane four times. The CD151/α3β1 integrin complex was found to localize at basolaterai surfaces of epithelial cells, where it was involved in cell-cell adhesion through promoting reorganisation of actin cytoskeleton Less

细胞与基底膜相互作用的分子机制进行了研究，重点是整合素介导的信号转导。主要成就概述如下。负责细胞粘附到基底膜上的主要整合素同种型，即，在非变性条件下将整联蛋白α3β1和α6β1纯化至均一。这些整合素的配体结合特异性通过固相结合测定法针对一组α亚基组成不同的层粘连蛋白同种型测定。我们的结果清楚地表明，含有α5链的层粘连蛋白-10是两种层粘连蛋白结合整合素的最优选配体.发现通过这些整联蛋白粘附到层粘连蛋白-10上的细胞选择性地激活Rho家族GTP酶的成员Rac，从而强烈促进板状伪足的形成和细胞运动性。层粘连蛋白-10上的Rac活化与p130 Cas的选择性酪氨酸磷酸化有关，而与FAK无关，如下： ...更多信息 d通过p130 Cas、CrkII和DOCK 180之间的三元复合物形成，后者是Rac的鸟嘌呤核苷酸交换因子。还发现细胞粘附到层粘连蛋白-10上激活PI 3 K-Akt通路，导致Akt的强烈激活。与Akt在细胞存活中的作用一致，层粘连蛋白-10上的细胞比纤连蛋白上的细胞对诱导的凋亡的抗性更强。通过，血清剥夺。有趣的是，细胞在纤连蛋白上的存活主要依赖于ERK通路，而不是PI 3 K-Akt通路.层粘连蛋白-8，主要层粘连蛋白亚型的内皮基底膜，纯化到同质的人胶质瘤细胞的条件培养基。与层粘连蛋白-10.5上的细胞粘附一样，层粘连蛋白-8上的细胞粘附严格依赖于整合素α6β1并优先激活Rac。结果表明，整合素α3β1与一个分子量为30 kD的蛋白质共纯化。这种30 kD的蛋白被鉴定为CD 151，是跨质膜四次的四跨膜蛋白家族的成员。发现CD 151/α3β1整联蛋白复合物定位于上皮细胞的基底层表面，在那里它通过促进肌动蛋白细胞骨架的重组参与细胞间粘附。