Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments
Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments
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DOI：
10.1073/pnas.0405973101
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发表时间：
2004-11-23
影响因子：
11.1
通讯作者：
Zhang, J
中科院分区：
文献类型：
--
作者：
DiPilato, LM;Cheng, XD;Zhang, J
第二信使cAMP通过其效应物调节许多细胞功能，例如cAMP依赖性蛋白激酶（PKA）和Epac（由cAMP直接激活的交换蛋白）。cAMP信号传导的空间和时间控制对于参与各种信号传导级联的细胞靶标的差异调节至关重要。为了研究区室化的cAMP信号传导，我们构建了荧光指示剂，通过在绿色荧光蛋白的青色和黄色突变体之间插入全长Epac 1来报告细胞内cAMP动力学和Epac激活。在活的哺乳动物细胞中，cAMP的升高降低了FRET，并使青色与黄色发射的比率增加了10-30%。这种反应可以通过去除cAMP升高剂来逆转，并通过突变负责cAMP结合的关键残基来消除。将报告基因靶向质膜（其中cAMP响应于β-肾上腺素能受体的活化而产生），揭示了膜处的cAMP响应比细胞质和线粒体中的更快。同时成像与靶向cAMP指示剂和PKA活性报告允许检测一个非常延迟的PKA反应后，在同一细胞的质膜上的cAMP的快速积累，尽管立即存在的cAMP池在核中。因此，cAMP动力学及其效应物的激活在体内时空上受到精确控制。
Second messenger cAMP regulates many cellular functions through its effectors, such as cAMP-dependent protein kinase (PKA) and Epac (exchange proteins directly activated by cAMP). Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. To investigate the compartmentalized cAMP signaling, we constructed fluorescent indicators that report intracellular cAMP dynamics and Epac activation by sandwiching the full-length Epac1 between cyan and yellow mutants of GFP. Elevations of cAMP decreased FRET and increased the ratio of cyan-to-yellow emissions by 10-30% in living mammalian cells. This response can be reversed by removing cAMP-elevating agents and abolished by mutating the critical residue responsible for cAMP binding. Targeting of the reporter to the plasma membrane, where cAMP is produced in response to the activation of beta-adrenergic receptor, revealed a faster cAMP response at the membrane than in the cytoplasm and mitochondria. Simultaneous imaging with targeted cAMP indicator and PKA activity reporter allowed the detection of a much delayed PKA response in the nucleus after the rapid accumulation of cAMP at the plasma membrane of the same cell, despite the immediate presence of a pool of cAMP in the nucleus. Thus, cAMP dynamics and the activation of its effectors are precisely controlled spatiotemporally in vivo.