Epac/cAMP-GEF, A Novel Intracellular cAMP Receptor

Epac/cAMP-GEF，一种新型细胞内 cAMP 受体

• 批准号: 6743684
• 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/6743684
• 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）： 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Rap 1。Epac的发现为cAMP介导的信号转导研究开辟了新的方向。特别地，除了PKA之外的第二种细胞内cAMP受体的发现表明，大量cAMP文献中描述的一些或甚至大多数cAMP作用并不像以前认为的那样通过单独激活PKA起作用。最近的研究还表明，Epac是一种多功能蛋白，能够介导cAMP的行动不同于PKA。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。具体而言，我们将：1）使用免疫荧光显微镜和GFP研究Epac的亚细胞靶向，特别是线粒体定位，及其对Epac的细胞功能的重要调节（绿色荧光蛋白）融合蛋白; 2）检验微管蛋白/微管，作为上游调节物或下游效应物，是一个真正的Epac细胞伴侣，通过检查Epac和微管蛋白/微管相互作用的生化和功能后果; 3）使用化学蛋白质足迹法和灵敏的MALDI-TOF技术，绘制与通过cAMP和Epac-Rap 1和Epac-微管蛋白相互作用激活Epac相关的构象变化，所述相互作用对Epac功能是重要的（基质辅助激光解吸/电离飞行时间）映射技术。 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是如何组装和协调的，以在分子和结构水平上介导cAMP信号传导。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的细胞调节作用是必不可少的。