Optical biosensor provides insights for bradykinin B2 receptor signaling in A431 cells
Optical biosensor provides insights for bradykinin B2 receptor signaling in A431 cells
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DOI：
10.1016/j.febslet.2005.10.019
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发表时间：
2005-11-21
期刊：
影响因子：
3.5
通讯作者：
Peng, JL
中科院分区：
文献类型：
--
作者：
Fang, Y;Li, GS;Peng, JL
蛋白质或蛋白质组合的空间和时间靶向对调节蛋白质相互作用的特异性和效率至关重要，因此决定了细胞信号传导和反应的时间和强度。由此产生的动态质量重分布可以通过无标记光学生物传感器来表现，并为研究细胞信号传递提供了一种新的、功能性的光学特征。在这里，我们应用这种技术，称为质量再分配细胞测定技术（MRCAT），研究A431细胞中缓激肽B-2受体的信号网络。利用MRCAT，在微孔板格式和活细胞中定量监测缓激肽介导的蛋白质和蛋白质组装的时空迁移。对缓激肽的饱和性，以及B-2特异性拮抗剂HOE140的特异性和剂量依赖性抑制，表明光学特征是B-2受体激活的直接结果。甲基- β -环糊精对胆固醇消耗的光学特征的敏感性表明，B-2受体信号传导依赖于脂筏的完整性；这些微结构域的破坏阻碍了B-2信号传导。用特定抑制剂调节几个重要的细胞内靶点表明，B-2受体激活至少通过双途径——G(s)和G(q)介导的信号传导导致信号传导。值得注意的是，这两种信号通路相互反向调节。包括蛋白激酶C、蛋白激酶A和表皮生长因子受体在内的几个关键下游靶点已被确定参与B-2信号传导。内吞作用和细胞骨架调节在B-2信号传导中的作用也得到了证实。(c) 2005年欧洲生化学会联合会。Elsevier B.V.版权所有。
The spatial and temporal targeting of proteins or protein assemblies to appropriate sites is crucial to regulate the specificity and efficiency of protein-protein interactions, thus dictating the timing and intensity of cell signaling and responses. The resultant dynamic mass redistribution could be manifested by label free optical biosensor, and lead to a novel and functional optical signature for studying cell signaling. Here we applied this technology, termed as mass redistribution cell assay technology (MRCAT), to study the signaling networks of bradykinin B-2 receptor in A431 cells. Using MRCAT, the spatial and temporal relocation of proteins and protein assemblies mediated by bradykinin was quantitatively monitored in microplate format and in live cells. The saturability to bradykinin, together with the specific and dose-dependent inhibition by a B-2 specific antagonist HOE140, suggested that the optical signature is a direct result of B-2 receptor activation. The sensitivity of the optical signature to cholesterol depletion by methyl-beta-cyclodextrin argued that B-2 receptor signaling is dependent on the integrity of lipid rafts; disruption of these microdomains hinders the B-2 signaling. Modulations of several important intracellular targets with specific inhibitors suggested that B-2 receptor activation results in signaling via at least dual pathways - G(s)- and G(q)-mediated signaling. Remarkably, the two signaling pathways counter-regulate each other. Several critical downstream targets including protein kinase C, protein kinase A, and epidermal growth factor receptor had been identified to involve in B-2 signaling. The roles of endocytosis and cytoskeleton modulation in B-2 signaling were also demonstrated. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.