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

研究了细胞-基底膜相互作用的分子机制，重点研究了整合素介导的信号转导。主要成就总结如下。在非变性条件下，整合素α3β1和α6β1等主要整合素同型体被纯化至均匀。这些整合素的配体结合特异性是通过固相结合试验确定的，它们与α亚基组成不同的一组层粘连蛋白同种异构体结合。我们的研究结果清楚地表明，含有α5链的层粘连蛋白-10是两种层粘连蛋白结合的整合素最优选的配体。细胞通过这些整合素粘附在层粘连蛋白-10上，选择性地激活Rho家族GTPases成员Rac，从而强烈促进板足的形成和细胞运动。Rac在laminin-10上的激活与p130Cas的选择性酪氨酸磷酸化有关，但与FAK无关，其次是p130Cas、CrkII和DOCK180之间形成的三联络合物，后者是Rac的鸟嘌呤核苷酸交换因子。细胞粘附在laminin-10上也被发现激活PI3K-Akt通路，导致Akt的强烈活化，与Akt在细胞存活中的作用一致，laminin-10上的细胞比纤维连接蛋白上的细胞对诱导的凋亡具有更强的抵抗力。通过血清剥夺。有趣的是，纤维连接蛋白作用下的细胞存活主要依赖于ERK通路，而不依赖于PI3K-Akt通路。从人胶质瘤细胞的条件培养基中纯化了内皮基膜中主要的层粘连蛋白-8。细胞对lamin8的粘附严格依赖于整合素α6β1和优先激活Rac，与细胞对lamin10.5的粘附情况相同。发现一个30 kD的蛋白与整合素α3β1共凝。这个30 kD的蛋白被鉴定为CD151，是横跨质膜四次的四跨蛋白家族的成员。CD151/α3β1整合素复合物被发现定位于上皮细胞的基底侧表面，通过促进肌动蛋白细胞骨架Less的重组参与细胞-细胞粘附

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