Mechanisms of Compartmentalized cAMP Signaling

描述（由申请人提供）： 环状 3', 5'-单磷酸腺苷 (cAMP) 是经典的第二信使，调节许多不同的细胞功能。尽管我们对 cAMP 和 cAMP 依赖性蛋白激酶 (PKA) 了解很多，但我们对 cAMP 信号的空间和时间性质以及特异性耦合 cAMP 及其效应物的分子机制（包括 PKA 和最近发现的由 cAMP 直接激活的交换蛋白 (Epac)）的理解仍然存在很大差距。我们研究的总体目标是阐明 cAMP 区室化在实现 cAMP 信号传导高特异性方面的机制和功能意义。具体目标是： 1) 进一步开发基因编码的 cAMP 指示剂，并表征由跨膜和可溶性腺苷酸环化酶 (AC) 产生的不同 cAMP 细胞池。这一拟议目标建立在使用该实验室最近开发的荧光 cAMP 指示剂获得的初步数据的基础上，并测试了 AC 和磷酸二酯酶在建立不同 cAMP 池中的参与情况。还将设计新一代指标。 2) 鉴定线粒体cAMP的功能效应子。荧光 cAMP 指示剂成像显示，β 肾上腺素受体激活后，cAMP 在线粒体中快速积累，但其功能作用仍不清楚。 Epac 将作为线粒体 cAMP 的功能效应器，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之间的联系以及胰岛素对此联系的影响。活细胞荧光成像、生化测定和药理学操作将用于检验长期胰岛素预处理破坏 β-AR 和 PKA 之间的联系的假设。这里提出的研究应该有助于更好地理解将 cAMP 的产生、降解和功能耦合与效应器区分开的分子机制。 cAMP 信号传导受损对肥胖和 II 型糖尿病等临床病症具有广泛影响，特别是因为它与正常脂肪细胞代谢有关。对 cAMP 信号传导特异性的机制理解对于制定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.