Mechanisms of Compartmentalized cAMP Signaling

环3‘，5’-腺苷一磷酸(CAMP)是经典的第二信使，调节多种不同的 细胞功能。虽然关于cAMP和cAMP依赖的蛋白激酶(PKA)已经了解了很多， 在我们对cAMP信号的空间和时间性质的理解以及 特异性偶联cAMP及其效应物的分子机制，包括PKA和最近 发现由cAMP直接激活的交换蛋白(EPAC)。我们研究的总体目标是 阐明cAMP区隔在实现高密度脂蛋白中的机制和功能意义 CAMP信号的特异性。 具体目标是：1)进一步开发遗传编码的cAMP指标，并确定 跨膜和可溶性腺苷环化酶(AC)产生不同的cAMP细胞池。这 拟议的目标建立在最近使用荧光cAMP指示器获得的初步数据基础上 在本实验室开发，并测试AC和磷酸二酯酶参与建立不同的 露营地的池塘。新一代指标也将被设计出来。2)确定功能效应器 线粒体cAMP。荧光cAMP指示剂成像显示cAMP在 线粒体激活后的β肾上腺素能受体，其功能作用尚不清楚。 EPAC将被检测为线粒体cAMP的功能效应器，使用荧光成像，蛋白质 工程学、化学生物学和生化技术。3)分析测试版之间的联系 肾上腺素能受体(β-AR)和PKA以及胰岛素对这种联系的影响。活细胞荧光 成像、生化分析和药理操作将被用来检验这一假设 慢性胰岛素预处理破坏了β-AR和PKA之间的联系。 这里提出的研究应该会使人们更好地理解 将cAMP的产生、降解和功能偶联与效应器区分开来。受损的 CAMP信号对肥胖和II型糖尿病等临床疾病有广泛的影响 糖尿病，特别是当它与正常的脂肪细胞代谢有关时。对夏令营的机械理解 信号的特异性对于开发针对这些临床疾病的治疗策略至关重要。

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.