Novel single chain cAMP sensors for receptor-induced signal propagation
Novel single chain cAMP sensors for receptor-induced signal propagation
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DOI：
10.1074/jbc.c400302200
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发表时间：
2004-09-03
影响因子：
4.8
通讯作者：
Lohse, MJ
中科院分区：
文献类型：
--
作者：
Nikolaev, VO;Bünemann, M;Lohse, MJ
cAMP是许多G蛋白偶联受体的通用第二信使，并调节多种细胞事件。cAMP通过cAMP依赖性蛋白激酶（PKA）、cAMP门控离子通道和由cAMP直接激活的交换蛋白的两种同种型（Epac）发挥其作用。在这里，我们报告的环腺苷酸的Epac和PKA的cAMP结合结构域的基础上，新的荧光指示剂的发展。荧光共振能量转移之间的变体的绿色荧光蛋白（增强的青色荧光蛋白和增强的黄色荧光蛋白）直接融合的cAMP结合结构域用于分析空间和时间方面的cAMP信号在不同的细胞。与以前开发的基于PKA的指示剂相比，这些探针仅由缺乏协同性、催化特性和与其他蛋白质的相互作用的单个结合位点组成，从而使我们能够容易地对游离细胞内cAMP和快速信号传导事件进行成像。观察到快速β-肾上腺素能受体诱导的cAMP信号以高速（约40 μ m/s）在海马神经元和腹腔巨噬细胞的整个细胞体中行进。所开发的指标可广泛应用于研究cAMP及其生理作用和时空调控。
cAMP is a universal second messenger of many G-protein-coupled receptors and regulates a wide variety of cellular events. cAMP exerts its effects via cAMP-dependent protein kinase (PKA), cAMP-gated ion channels, and two isoforms of exchange protein directly activated by cAMP ( Epac). Here we report the development of novel fluorescent indicators for cAMP based on the cAMP-binding domains of Epac and PKA. Fluorescence resonance energy transfer between variants of green fluorescent protein ( enhanced cyan fluorescent protein and enhanced yellow fluorescent protein) fused directly to the cAMP-binding domains was used to analyze spatial and temporal aspects of cAMP-signaling in different cells. In contrast to previously developed PKA-based indicators, these probes are comprised of only a single binding site lacking cooperativity, catalytic properties, and interactions with other proteins and thereby allow us to easily image free intracellular cAMP and rapid signaling events. Rapid beta-adrenergic receptor-induced cAMP signals were observed to travel with high speed (approximate to 40 mum/s) throughout the entire cell body of hippocampal neurons and peritoneal macrophages. The developed indicators could be ubiquitously applied to studying cAMP, its physiological role and spatiotemporal regulation.