Mechanisms of Compartmentalized cAMP Signaling

描述（由申请人提供）：环3 '，5'-腺苷一磷酸（cAMP），经典的第二信使，调节许多不同的细胞功能。虽然已经了解了很多关于cAMP和cAMP依赖性蛋白激酶（PKA），仍然有很大的差距，在我们的理解的空间和时间性质的cAMP信号和分子机制，具体耦合cAMP及其效应，包括PKA和最近发现的交换蛋白直接激活cAMP（Epac）。我们的研究的总体目标是阐明cAMP区室化在实现cAMP信号传导的高度特异性中的机制和功能意义。具体目标是：1）进一步开发基因编码的cAMP指示剂，并表征跨膜和可溶性腺苷酸环化酶（AC）产生的cAMP的不同细胞池。这一拟议的目标建立在获得的初步数据与荧光cAMP指示剂，最近在本实验室开发和测试的参与AC和磷酸二酯酶在建立不同的池的cAMP。还将设计新一代指标。2)确定线粒体cAMP的功能效应物。用荧光cAMP指示剂成像显示，在β肾上腺素能受体激活后，cAMP在线粒体中快速积聚，其功能作用仍不清楚。将使用荧光成像、蛋白质工程、化学生物学和生物化学技术来测试Epac作为线粒体cAMP的功能效应器。3)分析β-肾上腺素能受体（β-AR）与蛋白激酶A（PKA）之间的联系及胰岛素对这种联系的影响。活细胞荧光成像、生化分析和药理学操作将用于检验β-AR和PKA之间的联系被慢性胰岛素预处理破坏的假设。这里提出的研究应导致更好地理解的分子机制，划分的生产，降解和功能耦合cAMP的效应。受损的cAMP信号传导对临床病症如肥胖症和II型糖尿病具有广泛的影响，特别是因为它涉及正常的脂肪细胞代谢。对cAMP信号传导特异性的机制理解对于开发这些临床病症的治疗策略至关重要。

DESCRIPTION (provided by applicant): Cyclic 3', 5'-adenosine monophosphate (cAMP), the classical second messenger, regulates many diverse cellular functions. Although much has been learned about cAMP and cAMP-dependent protein kinase (PKA), there are still large gaps in our understanding of the spatial and temporal nature of cAMP signals and the molecular mechanisms that specifically couple cAMP and its effectors, including PKA and recently discovered exchange proteins directly activated by cAMP (Epac). The overall goal of our research is to elucidate the mechanisms and functional significance of cAMP compartmentation in achieving high specificity in cAMP signaling. The specific aims are: 1) To further develop genetically encoded cAMP indicators and characterize the distinct cellular pools of cAMP generated by transmembrane and soluble adenylyl cyclases (AC). This proposed aim builds on preliminary data obtained with a fluorescent cAMP indicator that was recently developed in this laboratory and tests the involvement of AC and phosphodiesterase in establishing distinct pools of cAMP. A new generation of indicators will also be engineered. 2) To identify the functional effectors of mitochondrial cAMP. Imaging with the fluorescent cAMP indicator revealed rapid accumulation of cAMP in mitochondria following activation of beta adrenergic receptor, the functional role of which remains unknown. Epac will be tested as the functional effector of mitochondrial cAMP using fluorescent imaging, protein engineering, chemical biology and biochemical techniques. 3) To analyze the linkage between beta adrenergic receptor (beta-AR) and PKA and the effect of insulin on this linkage. Live-cell fluorescence imaging, biochemical assays, and pharmacological manipulation will be used to test the hypothesis that the linkage between beta-AR and PKA is disrupted by chronic insulin pretreatment. The studies proposed here should lead to a greater understanding of the molecular mechanisms that compartmentalize the production, degradation, and functional coupling of cAMP to the effectors. Impaired cAMP signaling has widespread implications for clinical conditions such as obesity and type II diabetes mellitus, particularly as it relates to normal adipocyte metabolism. A mechanistic understanding of cAMP signaling specificity is crucial to developing therapeutic strategies for these clinical conditions.