Mechanisms of Compartmentalized cAMP Signaling

区室化 cAMP 信号传导机制

• 批准号: 7167435
• 负责人: Jin Zhang
• 金额: $ 29.42万
• 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/7167435
• 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; Condition; 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; 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; Condition; 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

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.

描述（由申请人提供）：经典第二信使（CAMP）的环状3'，5'-腺苷（CAMP）调节许多不同的细胞功能。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以及特殊搭配营地及其效应子（包括PKA）的分子机制的理解仍然存在很大的差距，包括PKA，最近发现了CAMP直接激活的交换蛋白（EPAC）（EPAC）。我们研究的总体目标是阐明营地隔室在达到cAMP信号中高特异性方面的机制和功能意义。具体目的是：1）进一步开发遗传编码的cAMP指标，并表征由跨膜和可溶性腺苷循环酶（AC）产生的cAMP的独特细胞池。该拟议的目的是建立在最近在该实验室中开发的荧光cAMP指标获得的初步数据，并测试了AC和磷酸二酯酶在建立不同的CAMP池中的参与。新一代的指标也将进行设计。 2）确定线粒体cAPP的功能效应子。用荧光cAMP指标进行成像显示，在激活β肾上腺素能受体激活后，cAMP在线粒体中的迅速积累，其功能作用仍然未知。 EPAC将使用荧光成像，蛋白质工程，化学生物学和生化技术来测试线粒体cAMP的功能效应因子。 3）分析β肾上腺素能受体（Beta-AR）和PKA之间的联系以及胰岛素对这种链接的影响。活细胞荧光成像，生化测定和药理学操作将用于检验以下假设：βAR和PKA之间的连接受到慢性胰岛素预处理的破坏。这里提出的研究应更深入地了解将CAMP与效应子的产生，降解和功能耦合分散的分子机制。营地信号受损对肥胖症和II型糖尿病等临床状况具有广泛的影响，特别是因为它与正常的脂肪细胞代谢有关。对CAMP信号特异性的机械理解对于为这些临床状况制定治疗策略至关重要。