Analysis of Ras and Rap activation in living cells using fluorescent Ras binding domains
Analysis of Ras and Rap activation in living cells using fluorescent Ras binding domains
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DOI：
10.1016/j.ymeth.2005.05.022
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发表时间：
2005-10-01
期刊：
影响因子：
4.8
通讯作者：
Philips, MR
中科院分区：
文献类型：
--
作者：
Bivona, TG;Philips, MR
Ras GTP酶调节细胞的生长和分化，并受到无数刺激物的调节，包括生长因子、细胞因子、抗原和紫外线照射。Ras GTP酶是当GTP结合时有活性而当GDP结合时无活性的分子开关。这些GTP酶的信号传导能力需要GTP结合形式与下游效应物接合，这种相互作用仅发生在细胞膜的胞质表面上。Ras家族蛋白包括H-Ras、N-Ras、K-Ras和Rap 1。对这些分子的调节和信号传导特性的了解主要来自依赖于细胞刺激后制备的细胞提取物的体外研究。由于Ras GTP酶在多个细胞区室上表达，所述多个细胞区室包括质膜、来源于质膜的囊泡和其他内膜如ER和高尔基体，因此其空间分布如何调节信号传导的分析仍然未知。我们已经开发了荧光，GFP为基础的探针能够选择性地结合GTP结合的Ras或Rap 1在活细胞。我们已经使用这些记者检查的Ras和Rap 1在生长因子刺激过程中的细胞激活的网站。这些研究揭示了这些GTPases信号平台的新见解，并导致GTPases信号以区室化方式调节的假设。在这里，我们描述的Ras和Rap 1的荧光探针的设计和实施。(c)2005年爱思唯尔公司All rights reserved.
Ras GTPases regulate cellular growth and differentiation and are modulated by myriad stimuli including growth factors, cytokines, antigens, and UV irradiation. Ras GTPases are molecular switches that are active when GTP-bound and inactive when GDP-bound. The ability of these GTPases to signal requires that the GTP-bound form engage downstream effectors, interactions that occur only on the cytosolic surface of cellular membranes. Ras family proteins include H-Ras, N-Ras, K-Ras, and Rap1. Insight into the regulation and signaling properties of these molecules has come largely from in vitro studies relying on cellular extracts prepared following cellular stimulation. Since Ras GTPases are expressed on multiple cellular compartments that include the plasma membrane, vesicles derived from the plasma membrane, and other internal membranes such as the ER and Golgi complex, analysis of how their spatial distribution modulates signaling has remained unknown. We have developed fluorescent, GFP-based probes capable of selectively binding GTP-bound Ras or Rap1 in living cells. We have used these reporters to examine sites of cellular activation of Ras and Rap1 during growth factor stimulation. These studies have revealed new insights into the platforms from which these GTPases signal and have led to the hypothesis that GTPase signaling is modulated in a compartmentalized fashion. Here, we describe the design and implementation of fluorescent probes for Ras and Rap1. (c) 2005 Elsevier Inc. All rights reserved.