利用显性负模型研究整合素β3胞浆段RGT序列对信号转导的调控机制  

Analysis of the Regulatory Mechanisms of the RGT Sequence of Integrin β3 Cytoplasmic Tail in Signal Transduction by Using a Dominant Negative Model

作　　者：陶岚岚[1] 黄建松[1] 吕媛靖[1] 周玉兰[1] 崔雄鹰[1] 阮铮[1] 奚晓东[1] 

机构地区：[1]上海交通大学附属瑞金医院上海血液学研究所医学基因组国家重点实验室,上海200025

出　　处：《中国细胞生物学学报》2012年第2期135-145,共11页Chinese Journal of Cell Biology

基　　金：国家高技术研究发展计划(863)(No.2006AA02A245);国家自然科学基金(No.81070414);上海市科委国际合作项目(No.09410706800)资助项目~~

摘　　要：该研究探讨整合素β3胞浆段RGT序列在整合素信号转导中的作用。利用IL-2R胞外段和跨膜段(Tac)与整合素β3胞浆段全长或截短序列构建Tac-β3嵌合体,即Tac-β3和Tac-β3Δ759,使其分别在表达GPIbIX、整合素αIIbβ3的CHO细胞株(IbIX/IIbIIIa-CHO细胞株)中稳定表达(即IbIX/IIbIIIa-CHO/Tac-β3、IbIX/IIbIIIa-CHO/Tac-β3Δ759细胞株)。利用竞争酶联免疫法对Tac-β3嵌合体进行定量,观察IbIX/IIbIIIa-CHO/Tac-β3、IbIX/IIbIIIa-CHO/Tac-β3Δ759细胞株在固相化纤维蛋白原上的伸展情况(代表外向内的信号转导);Co-IP研究Tac-β3嵌合体、β3与下游信号分子Src的结合情况。结果发现:竞争酶联免疫法证实了两个细胞株的Tac-β3嵌合体表达都高于野生型β3,保证了Tac-β3嵌合体对内源性β3在结合胞内分子中的显性负效应;IbIX/IIbIIIa-CHO/Tac-β3细胞株在固相化纤维蛋白原上的黏附伸展能力受到抑制,而IbIX/IIbIIIa-CHO/Tac-β3Δ759细胞株在固相化纤维蛋白原上的黏附伸展能力并未受到明显影响。Co-IP结果显示:Tac-β3能结合内源性Src,而胞浆段RGT序列缺失的Tac-β3Δ759则不与内源性Src结合。选择性地破坏Tac-β3的Src结合能力即足以去除其对外向内信号转导的显性负效应,从而表明:整合素β3尾端RGT序列与Src的相互作用在这一信号转导通路中起决定性的作用。This study purposed to investigate the molecular mechanisms of the RGT sequence of integrin β3 cytoplasmic tail in signal transduction by using a dominant negative cell model.Constructs encoding a chimeric protein composed of the extracellular and transmembrane domains of interleukin-2 receptor(Tac) and the full length or RGT-truncated β3 intracellular domain were expressed in CHO cells expressing GPIbIX and integrin αIIbβ3(IbIX/IIbIIIa-CHO cell line) to establish the stable IbIX/IIbIIIa-CHO/Tac-β3 and IbIX/IIbIIIa-CHO/Tac-β3?759 cell lines.Competitive ELISA was performed to quantify the expression level of Tac-β3 chimeras in the dominant negative cell lines.Spreading and stable adhesion of the IbIX/IIbIIIa-CHO/Tac-β3 and IbIX/IIbIIIa-CHO/Tac-β3?759 cells on immobilized fibrinogen(Fg) were examined to evaluate the transduction of outside-in signals.The interaction of Tac-β3 chimeras and endogenous β3 with Src kinase was simultaneously analyzed with Co-IP.Competitive ELISA showed that the expression level of Tac-β3 chimeras was substantially higher than that of endogenous β3 in both IbIX/IIbIIIa-CHO/Tac-β3 and IbIX/IIbIIIa-CHO/Tac-β3?759 cell lines that ensures the dominant negative effect of the Tac-β3 chimeras over the endogenous wild type β3 in binding intracellular molecules.In deed,the ability of IbIX/IIbIIIa-CHO/Tac-β3 cells in spreading and stable adhesion on immobilized Fg was effectively inhibited,while that of IbIX/IIbIIIa-CHO/Tac-β3?759 cells was not affected.Co-IP results demonstrate that the Tac-β3 chimeras competed with the endogenous integrin β3 in binding Src,while Tac-β3?759 lacking the RGT sequence at the C terminal of β3 cytoplasmic tail lost this ability.Selective disruption of Src-binding capacity of Tac-β3 is sufficient to eliminate its dominant negative effect on outside-in signaling suggesting that the interaction of the RGT sequence at the integrin β3 tail with Src kinase is crucial for this signaling pathway.

关 键 词：显性负 整合素αIIbβ3 竞争酶联免疫测定 蛋白质相互作用 信号转导 

分 类 号：R346[医药卫生—基础医学]