MECHANISMS AND VARIATIONS OF INTEGRIN-MEDIATED SIGNAL TRANSDUCTION

整合素是纤维连接蛋白和层粘连蛋白等细胞外基质黏附糖蛋白的主要受体。细胞外配体与整合素的结合是通过连接蛋白(如粘着斑蛋白、PXLIN、张力蛋白、pl30Cas)的酪氨酸磷酸化，然后激活Rho家族的小GTP结合蛋白(如Rac、Rho、CDC42)和ras/MAP激酶信号通路来传递信号。在这项研究中，我们集中精力研究了整合素与层粘连蛋白(LNS)的相互作用，层粘连蛋白是基底膜的主要黏附蛋白。本研究的主要发现如下：(1)LN-8的提纯与鉴定：利用RTPCR法筛选了5条不同的LNα链在35种以上的人细胞系中的表达。在5条LNα链中，T98G胶质瘤细胞仅表达α4链。我们还在免疫印迹上制备了识别α4链的单抗，发现该抗体在培养的…中识别出600kDa的条带在非还原条件下，T98G细胞上清液较多。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T98G细胞的黏附作用弱于LN-5和LN-10/11，而黏附活性与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的细胞骨架调控：最近，我们成功地纯化了含有完整α5链的LN-10/11(Kikkawa等人)。J.Biol.化学。247、15854-15859、1998年)。我们研究了黏附在LN-10/11上是否会导致应力纤维和局灶性黏连的形成，这是黏附在纤维连接蛋白包裹的底物上的细胞表型的标志。尽管LN-10/11具有很强的细胞黏附活性，但它未能在黏附于LN-10/11的细胞中诱导应力纤维或灶性黏附。我们还发现，在LN-10/11上未检测到Rho的激活，这与其未能诱导应力纤维和灶性黏附一致。(3)与整合素α3β1紧密相关的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，并从人胎盘中纯化了整合素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 0kDa/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。我们还制备了两种识别30 kDa蛋白的单抗，根据它们与CD151基因转染的NIH3T3细胞的反应性，证实了这两种抗体可以识别CD151。这些单抗在CD151的生理功能研究中具有一定的应用价值。较少

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