Epac/cAMP-GEF, A Novel Intracellular cAMP Receptor

Epac/cAMP-GEF，一种新型细胞内 cAMP 受体

• 批准号: 6617581
• 负责人: XIAODONG CHENG
• 金额: $ 27.12万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6617581
• 关键词: chemical fingerprinting; clinical research; conformation; cyclic AMP; cyclic AMP receptors; fluorescence microscopy; green fluorescent proteins; guanine nucleotide exchange factors; immunocytochemistry; matrix assisted laser desorption ionization; mitochondria; protein binding; protein localization; protein protein interaction; receptor binding; tubulin

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.

描述（由申请人提供）： 由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）是一种新型的细胞内cAMP受体，它直接激活Ras样小G蛋白Rap1。 Epac 的发现为 cAMP 介导的信号传导研究开辟了新的维度。特别是，除了 PKA 之外还发现了第二种细胞内 cAMP 受体，这表明大量 cAMP 文献中描述的一些甚至大部分 cAMP 作用并不像以前认为的那样通过单独激活 PKA 来发挥作用。最近的研究还表明，Epac 是一种多功能蛋白，能够介导与 PKA 不同的 cAMP 作用。该提案的主要目标是剖析 cAMP 信号通路，特别强调新型 cAMP 传感器蛋白 Epac 的结构和功能。具体来说，我们将： 1) 使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，特别是线粒体定位及其对 Epac 细胞功能重要的调节； 2) 通过检查 Epac 和微管蛋白/微管相互作用的生化和功能后果，检验微管蛋白/微管作为上游调节器或下游效应器是真正的 Epac 细胞伙伴的假设； 3) 使用化学蛋白质足迹和敏感的 MALDI-TOF（基质辅助激光解吸/电离时间）绘制与 cAMP 和 Epac-Rap1 以及 Epac-微管蛋白相互作用激活 Epac 相关的构象变化，这对 Epac 功能很重要-飞行）测绘技术。 我们的长期目标是通过剖析 Epac 介导的细胞和生化相互作用，了解 cAMP 结合和鸟嘌呤核苷酸交换活性的不同功能如何组装和协调，以在分子和结构水平上介导 cAMP 信号传导。这些拟议的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至关重要。