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）： 通过CAMP（EPAC）或CAMP激活的鸟嘌呤核苷酸交换因子（Camp-GEF）直接激活的交换蛋白是一种新型的细胞内营受体，它直接激活RAP1，一种RAS（一种RAS样的小G蛋白）。 EPAC的发现在CAMP介导的信号传导的研究中开辟了一个新的维度。特别是，除了PKA之外，发现第二个细胞内营受体的发现表明，庞大的营地文献中描述的一些或什至大多数营地行动并不能单独激活PKA，正如先前所认为的那样。最近的研究还表明，EPAC是一种多功能蛋白，能够与PKA不同地介导营地动作。该提案的主要目标是剖析营地信号途径，特别强调了新型营地传感器蛋白EPAC的结构和功能。具体而言，我们将：1）研究EPAC的亚细胞靶向，尤其是线粒体定位及其调节，对于使用免疫荧光显微镜和GFP（绿色荧光蛋白）融合蛋白的EPAC细胞功能很重要； 2）检验以下假设：通过检查EPAC和小管蛋白/微管相互作用的生化和功能后果，通过起作用是上游调节剂或下游效应子，是真正的EPAC细胞伴侣。 3）绘制与CAMP和EPAC-RAP1和EPAC-微管蛋白相互作用相关的构象变化，这些相互作用对于使用化学蛋白足迹和敏感的MALDI-TOF（基质辅助激光液/电离时间）对EPAC功能很重要 - 飞行）映射技术。 我们的长期目标是了解cAMP结合和鸟嘌呤核苷酸交换活性的不同功能如何组装并协调以通过解剖EPAC介导的细胞和生化相互作用来介导分子和结构水平的cAMP信号传导。这些提出的研究对于进一步了解cAMP信号通路并阐明了在正常和病理状态下的细胞调节中的cAMP的作用至关重要。