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MECHANISMS AND VARIATIONS OF INTEGRIN-MEDIATED SIGNAL TRANSDUCTION

整合素介导的信号转导的机制和变化

基本信息

批准号：
10680624
负责人：
SEKIGUCHI Kiyotoshi
金额：
$ 2.11万
依托单位：
OSAKA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

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

整合素是细胞外基质粘附糖蛋白如纤连蛋白和层粘连蛋白的主要受体。细胞外配体与整联蛋白的结合通过衔接蛋白（如粘着斑激酶、桩蛋白、张力蛋白、pl 30 Cas）的酪氨酸磷酸化，随后通过小GTP结合蛋白的rho家族（即rac、rho、cdc 42）和ras/MAP激酶信号传导级联的活化来转导信号。在这项研究中，我们集中精力在整合素与层粘连蛋白（LN），主要的基底膜粘附蛋白的相互作用。这项研究的主要结果如下。(1)LN-8的纯化和鉴定：我们通过RT-PCR在超过35个人细胞系中筛选了五种不同LN α链的表达。T98 G胶质瘤细胞仅表达5条LNα链中的α4链。我们还制备了识别α4链的单克隆抗体，并发现该抗体在培养物中识别600 kDa条带 ...更多信息 T98 G细胞的上清液在非还原条件下。我们大规模培养T98 G细胞，收获用过的培养基（约4升），通过硫酸铵沉淀浓缩，然后进行凝胶过滤和使用抗LN β1单克隆抗体4F 5的免疫亲和层析。在非还原条件下，所得蛋白在SDS-PAGE上明显是均一的，并且由于其由α4/β1/γ 1链组成，因此被鉴定为LN-8。纯化的LN-8在介导T98 G细胞粘附方面不如LN-5和LN-10/11有效，而在其细胞粘附活性方面与LN-1相当。细胞与LN-8的粘附是通过整合素α6β 1和α3β1介导的，这两种整合素是两种主要的LN结合整合素。(2)对LN-10/11的细胞骨架调节：最近，我们成功地纯化了含有完整形式的α5链的LN-10/11（Kikkawa等人，J. Biol. Chem. 247，15854-15859，1998）。我们检查了粘附到LN-10/11上是否可以诱导应力纤维和局灶性粘附的形成，这是粘附到纤连蛋白包被的基底上的细胞表型的标志。尽管其非常有效的细胞粘附活性，LN-10/11未能诱导应力纤维或局灶性粘附在LN-10/11粘附的细胞。我们还发现，在LN 10/11上未检测到rho的激活，这与其诱导应力纤维和局灶性粘连的失败一致。(3)与整合素α3β1紧密结合的30 kDa蛋白质作为CD 151的鉴定：我们制备了针对整合素α3亚基胞质结构域的多克隆抗体，并从人胎盘中纯化了整合素α3β1。由此纯化的整联蛋白α3β1在SDS-PAGE上产生三个主要条带，即对应于α3和β1链的150 kDa/120 kDa条带和意外的30 kDa条带。使用Hitoshi Hasegawa博士（Ehime University Medical School）提供的抗CD 151抗体，通过免疫沉淀，将30 kDa条带鉴定为CD 151，跨膜4超家族蛋白之一。我们还制备了两种均识别30 kDa蛋白质的单克隆抗体，并且基于其与用CD 151 cDNA转染的NIH 3 T3细胞的反应性，证实所述抗体识别CD 151。这些单克隆抗体在研究CD 151的生理功能方面有一定的应用价值。少