Mechanisms of Compartmentalized cAMP Signaling

环磷酸腺苷（cAMP），经典的第二信使，调节许多不同的 细胞功能。尽管已经对cAMP和cAMP依赖性蛋白激酶（PKA）有了很多了解， 在我们对cAMP信号的空间和时间性质的理解方面仍然存在很大的差距， 特异性偶联cAMP及其效应物的分子机制，包括PKA和最近 发现了由cAMP直接激活的交换蛋白（Epac）。我们研究的总体目标是 阐明了cAMP区室化在实现高血压中的机制和功能意义。 cAMP信号传导的特异性。 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cAMP的不同细胞池。这 提出的目标建立在荧光cAMP指示剂获得的初步数据基础上， 在这个实验室开发和测试AC和磷酸二酯酶在建立不同的参与， cAMP池。还将设计新一代指标。2)识别功能效应器 线粒体cAMP。用荧光cAMP指示剂成像显示cAMP在 在β肾上腺素能受体激活后，线粒体的功能作用仍然未知。 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、化学生物学和生物化学技术。3)为了分析贝塔系数和 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，生化分析和药理学操作将被用来测试的假设， β-AR和PKA之间的连接被慢性胰岛素预处理破坏。 这里提出的研究应该导致更好地理解分子机制， 区室化cAMP的产生、降解和与效应物的功能性偶联。受损 cAMP信号传导对肥胖和II型糖尿病等临床病症具有广泛的影响 尤其是当它涉及正常脂肪细胞代谢时。对cAMP的机制性理解 信号特异性对于开发这些临床病症的治疗策略是至关重要的。

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.