Mechanisms of Compartmentalized cAMP Signaling

描述（申请人提供）：环3′，5′-腺苷单磷酸（cAMP），经典的第二信使，调节多种细胞功能。尽管我们对cAMP和cAMP依赖性蛋白激酶（PKA）已经了解了很多，但我们对cAMP信号的时空性质以及cAMP及其效应物特异性偶联的分子机制的理解仍然存在很大差距，包括PKA和最近发现的由cAMP直接激活的交换蛋白（Epac）。我们研究的总体目标是阐明cAMP区隔在实现cAMP信号高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指标，并表征由跨膜和可溶性腺苷酸环化酶（AC）产生的不同cAMP细胞池。该实验室最近开发了一种荧光cAMP指示剂，并测试了AC和磷酸二酯酶在建立不同cAMP池中的作用。新一代指标也将被设计出来。2)鉴定线粒体cAMP的功能效应物。荧光cAMP成像显示，β肾上腺素能受体激活后，线粒体中cAMP快速积累，其功能作用尚不清楚。Epac将作为线粒体cAMP的功能效应物，使用荧光成像、蛋白质工程、化学生物学和生化技术进行测试。3)分析β -肾上腺素能受体（β - ar）与PKA的联动以及胰岛素对该联动的影响。活细胞荧光成像、生化分析和药理学操作将用于验证β - ar和PKA之间的联系被慢性胰岛素预处理破坏的假设。这里提出的研究应该导致对分子机制的更深入的了解，这些分子机制划分了cAMP的产生、降解和与效应物的功能耦合。cAMP信号传导受损对肥胖和II型糖尿病等临床疾病具有广泛的影响，特别是当它与正常的脂肪细胞代谢有关时。了解cAMP信号特异性的机制对于制定针对这些临床疾病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.