Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral.
Single-molecule fluorescence imaging of RalGDS on cell surfaces during signal transduction from Ras to Ral.
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DOI：
10.2142/biophysico.14.0_75
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发表时间：
2017
影响因子：
1.5
通讯作者：
Sako Y
中科院分区：
文献类型：
--
作者：
Yoshizawa R;Umeki N;Yanagawa M;Murata M;Sako Y
RalGDS是Ras效应子之一，并作为小G蛋白Ral的鸟嘌呤核苷酸交换因子发挥作用，其调节膜运输和细胞骨架重塑。RalGDS从细胞质到质膜的易位是Ral激活所必需的。在这项研究中，为了了解Ras-Ral信号传导的机制，我们对活HeLa细胞质膜上的RalGDS及其功能结构域（RBD和REMCDC）进行了单分子荧光分析。在EGF刺激细胞诱导Ras活化后，在质膜上观察到RalGDS和RBD分子密度增加，但未观察到REMCDC，这表明RalGDS的易位涉及GTP结合的活性形式的Ras和RalGDS的RBD之间的相互作用。而RBD在增加RalGDS和质膜之间的结合速率常数中发挥了重要作用，REMCDC结构域影响从膜的解离速率常数，其在Ras激活或Ral过表达后降低。Ras的Y 64残基和RalGDS分子簇参与了这种还原。从这些发现中，我们推断Ras活化不仅增加RalGDS的细胞表面密度，而且通过RalGDS的结构变化和/或Ral的积累以及GTP-Ras/RalGDS簇主动刺激RalGDS-Ral相互作用，以诱导Ral的完全活化。
RalGDS is one of the Ras effectors and functions as a guanine nucleotide exchange factor for the small G-protein, Ral, which regulates membrane trafficking and cytoskeletal remodeling. The translocation of RalGDS from the cytoplasm to the plasma membrane is required for Ral activation. In this study, to understand the mechanism of Ras–Ral signaling we performed a single-molecule fluorescence analysis of RalGDS and its functional domains (RBD and REMCDC) on the plasma membranes of living HeLa cells. Increased molecular density of RalGDS and RBD, but not REMCDC, was observed on the plasma membrane after EGF stimulation of the cells to induce Ras activation, suggesting that the translocation of RalGDS involves an interaction between the GTP-bound active form of Ras and the RBD of RalGDS. Whereas the RBD played an important role in increasing the association rate constant between RalGDS and the plasma membrane, the REMCDC domain affected the dissociation rate constant from the membrane, which decreased after Ras activation or the hyperexpression of Ral. The Y64 residue of Ras and clusters of RalGDS molecules were involved in this reduction. From these findings, we infer that Ras activation not merely increases the cell-surface density of RalGDS, but actively stimulates the RalGDS–Ral interaction through a structural change in RalGDS and/or the accumulation of Ral, as well as the GTP–Ras/RalGDS clusters, to induce the full activation of Ral.