Epac/cAMP-GEF, A Novel Intracellular cAMP Receptor

描述(由申请人提供)： CAMP直接激活的交换蛋白(EPAC)或cAMP激活的鸟嘌呤核苷酸交换因子(cAMP-gef)是一种新的细胞内cAMP受体，直接激活RAS样小G蛋白Rap1。EPAC的发现为研究cAMP介导的信号通路开辟了一个新的领域。特别是，除了PKA之外，第二个细胞内cAMP受体的发现表明，在大量的cAMP文献中描述的一些甚至大多数cAMP作用并不像以前认为的那样仅通过激活PKA来起作用。最近的研究还表明，EPAC是一种多功能蛋白，能够介导不同于PKA的cAMP作用。这项建议的主要目的是剖析cAMP信号通路，特别是新的cAMP感受器蛋白EPAC的结构和功能。具体地说，我们将：1)利用免疫荧光显微镜和GFP(绿色荧光蛋白)融合蛋白研究EPAC的亚细胞靶向，特别是线粒体的定位，及其对EPAC细胞功能的重要调控；2)通过检测EPAC和微管/微管相互作用的生化和功能结果，检验微管/微管作为上游调节因子或下游效应分子是真正的EPAC细胞伙伴的假设；3)使用化学蛋白质足迹和灵敏的MALDI-TOF(基质辅助激光解吸/电离飞行时间)作图技术，绘制与cAMP激活EPAC以及EPAC-Rap1和EPAC-微管蛋白相互作用有关的构象变化，这些相互作用对EPAC的功能非常重要。 我们的长期目标是通过解剖EPAC介导的细胞和生化相互作用，了解cAMP结合和鸟核苷酸交换活性这两个不同的功能是如何组装和协调的，从而在分子和结构水平上介导cAMP信号。这些研究对于进一步了解cAMP信号通路和阐明cAMP在正常和病理状态下细胞调节中的作用是必不可少的。

DESCRIPTION (provided by applicant): Exchange protein directly activated by cAMP (Epac) or cAMP-activated guanine nucleotide exchange factor (cAMP-GEF) is a novel intracellular cAMP receptor, which directly activates Rap1, a Ras-like small G protein. The discovery of Epac opens up a new dimension in the study of cAMP-mediated signaling. In particular, the finding of a second intracellular cAMP receptor in addition to PKA suggests that some, or even the majority of cAMP actions described in the vast cAMP literature, do not act through the activation of PKA alone, as previously believed. Recent studies also suggest that Epac is a multifunctional protein that is capable of mediating cAMP actions differentially from PKA. The major goal of this proposal is to dissect the cAMP-signaling pathways, with particular emphasis on the structure and function of the novel cAMP sensor protein Epac. Specifically we will: 1) study subcellular targeting of Epac, particularly the mitochondrial localization, and its regulation that is important for Epac's cellular functions using immunofluorescence microscopy and GFP (green fluorescent protein) fusion protein; 2) test the hypothesis that tubulin/microtubule, acting either as an upstream regulator or downstream effector, is a bona fide Epac cellular partner by examining the biochemical and functional consequences of Epac and tubulin/microtubule interaction; 3) map the conformational changes associated with the activation of Epac by cAMP and Epac- Rap1 and Epac-tubulin interactions that are important to Epac functions using chemical protein footprinting and a sensitive MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mapping technique. Our long-term goal is to understand how the disparate functions of cAMP binding and guanine nucleotide exchange activity are assembled and coordinated to mediate cAMP signaling at the molecular and structural levels by dissecting Epac-mediated cellular and biochemical interactions. These proposed studies are essential for further understanding cAMP signaling pathways and elucidating the actions of cAMP in cellular regulation under normal and pathologic states.