Mechanisms of Compartmentalized cAMP Signaling

区室化 cAMP 信号传导机制

• 批准号: 7537192
• 负责人: Jin Zhang
• 金额: $ 28.87万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7537192
• 关键词: 2' -adenylic acid; 3T3-L1 Cells; Adenylate Cyclase; Adipocytes; Adrenergic Agents; Adrenergic Receptor; Apoptosis; Biochemical; Biological Assay; Biology; Caveolae; Cell Survival; Cell membrane; Cell physiology; Cells; Chemicals; Chronic; Clinical; Coupling; Cyclic AMP; Cytoplasm; Data; Diffusion; Embryo; Engineering; Generations; Goals; Guanosine; Human; Image; Insulin; Kidney; Knowledge; Laboratories; Lead; Learning; Life; Mammalian Cell; Measures; Metabolism; Mitochondria; Molecular; Monitor; Nature; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Pattern; Production; Protein Engineering; Protein Kinase; Proteins; Regulation; Research; Research Personnel; Role; Second Messenger Systems; Signal Transduction; Specificity; Techniques; Testing; Therapeutic; adrenergic; beta-adrenergic receptor; fluorescence imaging; genetic linkage analysis; innovation; phosphoric diester hydrolase; second messenger; spatiotemporal

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.

循环3'，5'-腺苷单磷酸盐（CAMP），经典的第二使者，调节了许多多样化 细胞功能。尽管关于营地和camp依赖性蛋白激酶（PKA）的了解很多，但 我们对营地信号的空间和时间性质的理解以及 分子机制，专门将CAMP及其效应子（包括PKA）和最近的分子机制机制 发现的交换蛋白直接被CAMP（EPAC）激活。我们研究的总体目标是 阐明营地隔室的机制和功能意义 营地信号的特异性。 具体目的是：1）进一步开发遗传编码的营地指标并表征 由跨膜和可溶性腺苷酸环化酶（AC）产生的cAMP的独特细胞池。这 拟议的目标建立在最近用荧光cAMP指标获得的初步数据的基础上 在该实验室中开发，并测试AC和磷酸二酯酶在建立不同的 营地池。新一代的指标也将进行设计。 2）识别功能效应子 线粒体营地。用荧光cAMP指示器进行成像显示cAMP在 激活β肾上腺素能受体后的线粒体，其功能作用仍然未知。 EPAC将使用荧光成像，蛋白 工程，化学生物学和生化技术。 3）分析beta之间的联系 肾上腺素能受体（β-AR）和PKA以及胰岛素对这种连接的影响。活细胞荧光 成像，生化测定和药理学操纵将用于检验以下假设。 beta-ar和PKA之间的连接受到慢性胰岛素预处理的破坏。 这里提出的研究应提出对分子机制的更多了解 将cAMP与效应子的生产，降解和功能耦合分开。受损 营地信号对肥胖和II型糖尿病等临床状况具有广泛的影响 Mellitus，特别是与正常的脂肪细胞代谢有关。对营地的机械理解 信号传导特异性对于制定这些临床条件的治疗策略至关重要。