SIGNAL TRANSDUCTION BY INTEGRIN-MATRIX INTERACTION

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，特别是Interinα3β1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的纯化与鉴定：我们通过RT-α方法筛选了5条不同的LN DNA链在超过35个人细胞系中的表达。在5条LNα链中，T98G胶质瘤细胞仅表达α4链。我们大规模培养T98G细胞，收集废液(约4L)，用硫酸铵沉淀浓缩，然后用抗层粘连蛋白β-1单抗4F5进行凝胶过滤和免疫亲和层析。在非还原条件下得到的蛋白在SDSPAGE上呈明显的均一状态，由α4/β1/γI链组成，经鉴定为LN-8。纯化的LN-8对…的黏附能力较弱T98G细胞的黏附活性高于LN-5和LN-10/11，与LN-1相当。细胞与LN-8的黏附是通过整合素α6β1和α3β1介导的。(2)对LN-10/11的细胞骨架调节：我们检测了黏附在LN-10/11上是否会诱导应力纤维和局灶性黏连的形成。尽管LN-10/11具有很强的细胞黏附活性，但它不能在黏附于LN-10/11的细胞中诱导应力纤维或局灶性黏附。我们还发现在LN-10/11上没有检测到Rho的激活，这与它不能诱导应力纤维和灶性黏附一致。(3)与整合素α3β1紧密结合的30 kDa蛋白质鉴定为CD151：我们制备了抗整合素α3亚单位胞浆结构域的多克隆抗体和从人胎盘中纯化的整合素α3β1。纯化的整合素α3β1在SDSPAGE上有3条主带，即对应于α3和β1链的15 0 kDa/12 0 kDa条带和一条意外的30 kDa条带。经长谷川博士提供的抗CD151抗体的免疫沉淀，30 kDa的条带被鉴定为跨膜4超家族蛋白之一的CD151。我们还制备了两个识别30 kDa蛋白的单抗，并根据它们与CD151基因转染的NIH3T3细胞的反应性证实了它们识别CD151的能力。这些单抗可用于研究CD151的生理功能。(4)LNα3链基因敲除小鼠的制备：我们成功地建立了缺失LNα3链基因的小鼠。这些小鼠在出生时死亡，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诱导稳定的黏附，这与结合性水泡和异常半桥粒的存在是一致的。我们还在表皮基底膜上检测到一个新的整合素α3β1配体，该配体在没有LN-5的情况下起作用。我们还在没有LN-5的情况下，在表皮基底膜上发现了一个新的整合素α3β1配体。我们还发现突变的上皮细胞存在存活缺陷，这种缺陷可以被外源性LN-5、胶原或抗α6β4抗体拯救，提示通过α6或β4整合素的信号转导足以维持存活。较少

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