MECHANISMS AND VARIATIONS OF INTEGRIN-MEDIATED SIGNAL TRANSDUCTION

整合素是细胞外基质黏附糖蛋白（如纤维连接蛋白和层粘连蛋白）的主要受体。细胞外配体与整合素的结合通过酪氨酸磷酸化连接蛋白（如focal adhesion Kinase， paxillin, tensin, pl30Cas）传递信号，随后激活rho家族的小gtp结合蛋白（如rac， rho, cdc42）和ras/MAP激酶信号级联反应。在这项研究中，我们将重点放在整合素与层粘连蛋白（LNs）的相互作用上，层粘连蛋白是主要的基底膜粘附蛋白。本研究的主要发现如下：(1) LN-8的纯化和鉴定：我们通过RT-PCR筛选了5种不同LN α链在超过35种人细胞系中的表达。T98G胶质瘤细胞在5条LNα链中只表达α4链。我们还在免疫印迹上制备了识别α4链的单克隆抗体，发现该抗体在非还原条件下培养的T98G细胞上清中鉴定了600 kDa的条带。我们对T98G细胞进行了大规模培养，并收获了用硫酸铵沉淀浓缩的培养基（约4升），然后使用抗ln β1单克隆抗体4F5进行凝胶过滤和免疫亲和层析。在非还原条件下，所得蛋白在SDS-PAGE上具有明显的同质性，由α4/β1/γi链组成，鉴定为LN-8。纯化后的LN-8对T98G细胞的粘附力低于LN-5和LN-10/11，其细胞粘附活性与LN-1相当。细胞粘附在LN-8上是通过两种主要的ln结合整合素α6β1和α3β1介导的。(2)细胞骨架对LN-10/11的调节：最近，我们成功纯化了含有完整α5链的LN-10/11 （Kikkawa etal .）。生物。化学，247,15854-15859,1998)。我们检测了粘附在LN-10/11上是否会诱导应力纤维和局灶性粘附的形成，这是粘附在纤维连接蛋白涂层底物上的细胞表型的标志。尽管LN-10/11具有很强的细胞粘附活性，但在粘附LN-10/11的细胞中，它不能诱导应力纤维或局灶性粘附。我们还发现在LN10/11上没有检测到rho的激活，这与它在诱导应力纤维和局灶粘连方面的失败一致。(3)鉴定与整合素α3β1紧密结合的30 kDa蛋白CD151：制备了针对整合素α3亚基细胞质结构域的多克隆抗体，并从人胎盘中纯化了整合素α3β1。纯化后的整联素α3β1在SDS-PAGE上有3个主要的条带，分别是α3和β1链对应的150 kda/120 kda条带和一个出乎意料的30 kda条带。利用爱媛大学医学院Hitoshi Hasegawa博士提供的抗CD 151抗体，通过免疫沉淀鉴定出30 kDa的条带为CD 151，是跨膜4超家族蛋白之一。我们还制备了两种识别30 kDa蛋白的单克隆抗体，根据抗体与转染CD 151 cDNA的NIH3T3细胞的反应性，证实抗体识别CD 151。这些单克隆抗体将在cd151的生理功能研究中发挥重要作用。少

Integrins are the major receptors for the extracellular matrix adhesive glycoproteins such as fibronectins and laminins. Binding of extracellular ligands to integrins transduces signals through tyrosine phosphorylation of adaptor proteins (such as focal adhesion Kinase, paxillin, tensin, pl30Cas) followed by activation of the rho family of small GTP-binding proteins (i.e. rac, rho, cdc42) and ras/MAP kinase signaling cascades. In this investigation, we focused our efforts on the interaction of integrins with laminins (LNs), the major basement membrane adhesive proteins. The major findings of this study are as follows.(1) Puification and Characterization of LN-8 : We screened the expression of five different LN α chains in more than 35 human cell lines by RT-PCR. The T98G glioma cells were found to express only α4 chain among the five LNα chains. We also produced a monoclonal antibody recognizing α4 chain on immunoblots and found that the antibody identified 600 kDa band in the culture … More supernatants of T98G cells under nonreducing conditions. 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 puified LN-8 was less potent in mediating adhesion 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 α6β1and α3β1, two major LN-binding integrins.(2) Cytoskeletal Modulation on LN-10/11 : Recently, we have succeeded in purifying LN-10/11 containing the α5 chain in its intact form (Kikkawa et al. J. Biol. Chem. 247, 15854-15859, 1998). We examined whether adhesion onto LN-10/11 could induce formation of stress fibers and focal adhesions, the hallmark of phenotypes of cells adhered onto fibronectin-coated substrates. 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 LN10/11, 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 CD 151, one of the transmembrane 4 superfamily proteins, by immunoprecipitation using the anti-CD 151 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 CD 151 based on their reactivity with the NIH3T3 cells transfected with the CD 151 cDNA. These monoclonal antilbodies should prove to be useful in the study of physiological funcions of CD 151. Less