Molecular signal transduction of cAMP compartments

cAMP 区室的分子信号转导

• 批准号: 10218196
• 负责人: RENNOLDS S OSTROM
• 金额: $ 30.04万
• 依托单位: CHAPMAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10218196
• 关键词: A kinase anchoring protein; Adenylate Cyclase; Adverse effects; Asthma; Biochemical; Biological Models; Biotin; Buffers; Cataloging; Cell Differentiation process; Cell membrane; Cell model; Cell physiology; Cells; Chronic Obstructive Airway Disease; Complex; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Diffusion; Disease; Elements; Enzymes; Event; Fibroblasts; Fibrosis; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; Goals; Heart failure; Hormone Receptor; Human; Hypertension; Individual; Kinetics; Label; Lead; Ligase; Lipids; Locales; Location; Measures; Membrane Microdomains; Methods; Molecular; Monitor; Nature; Neurotransmitter Receptor; Pharmaceutical Preparations; Physiological; Play; Positioning Attribute; Production; Protein Isoforms; Proteins; Proteomics; Receptor Signaling; Reporting; Role; Second Messenger Systems; Shapes; Signal Transduction; Signaling Protein; Small Interfering RNA; Smooth Muscle Myocytes; Stroke; Study models; Techniques; Therapeutic; Time; Vascular Diseases; Work; base; design; fluorophore; innovation; interdisciplinary approach; knock-down; member; mutant; novel; novel therapeutics; phosphoproteomics; phosphoric diester hydrolase; prevent; public health relevance; receptor; respiratory smooth muscle; response; scaffold; sensor; sensor technology; tool

A large number of G protein coupled receptors (GPCR) utilize cAMP as their second messenger to alter cell function. In fact, in the same cell several different GPCR can increase cAMP, leading to the question of how the cell interprets the signals from these receptors differently. The concept of cAMP compartmentation, where the second messenger is not generated uniformly throughout the cell, is readily accepted yet poorly understood. The enzymes that synthesize cAMP, adenylyl cyclases (ACs), are not uniformly distributed through the plasma membrane. Furthermore, GPCR can preferentially couple to certain AC isoforms due to colocalization in lipid rafts or non-raft domains. While we have made progress in understanding how specific receptors can couple to different ACs, little progress has been made in defining the compartments of cAMP inside cells and how cellular responses can be modified by different pools of cAMP. Commonly used cell models are de-differentiated and lack highly compartmentized cAMP pools. However, we have defined two clear cAMP signaling compartments in primary human airway smooth muscle (HASM) cells. The goal of this project is to characterize the key regulatory components, PDEs and AKAPs, in these two cAMP compartments and to discover novel protein members of signaling complexes therein. We will use siRNA to knockdown individual PDEs and AKAPs then measure localized cAMP signals via novel fluorescent sensors. We have defined the phosphoproteomic signatures of each cAMP compartment using quantitative phosphoproteomics, so will leverage these signatures to infer roles for individual PDEs or AKAPs following knockdown. State-of-the- art spectroscopic methods will directly assess the diffusion of cAMP in cells. Finally, we will use biotin proximity labeling to identify AC-interacting proteins in both HASM and less well differentiated HEK-293 cells. This project proposes innovative, multidisciplinary approaches to define the components responsible for establishing and maintaining cAMP signaling compartments. Our findings will have broad applicability due to the fundamental nature of cAMP signaling, but will also have direct relevance to asthma and COPD therapy.

大量G蛋白偶联受体（G protein coupled receptor，GPCR）以cAMP为第二信使，通过调节细胞内cAMP的水平， 功能事实上，在同一个细胞中，几种不同的GPCR可以增加cAMP，这导致了一个问题，即如何增加cAMP。 细胞对来自这些受体的信号有不同的解释。cAMP区室化的概念，其中 第二信使不是在整个细胞中均匀产生的， 明白合成cAMP的酶，腺苷酸环化酶（AC），并不均匀地分布在整个细胞中。 质膜。此外，GPCR可以优先与某些AC同种型偶联，这是由于 共定位在脂筏或非筏结构域。虽然我们已经在了解如何具体 尽管cAMP受体可以与不同的AC偶联，但在确定cAMP的区室方面几乎没有进展 以及不同的cAMP池如何改变细胞反应。常用的细胞 模型是去分化的，缺乏高度区室化的cAMP库。我们定义了两个 在原代人气道平滑肌（HASM）细胞中清除cAMP信号传导区室。这个目标 项目是表征这两个cAMP隔室中的关键调节组分PDE和AKAP 并发现其中信号传导复合物的新蛋白质成员。我们将使用siRNA敲除 然后，个体PDE和AKAP通过新型荧光传感器测量局部cAMP信号。我们有 使用定量磷酸蛋白质组学定义了每个cAMP区室的磷酸蛋白质组学特征， 因此将利用这些特征来推断敲除后个体PDE或AKAP的作用。国家 现有的光谱方法将直接评估细胞中cAMP的扩散。最后，我们将使用生物素邻近 标记以鉴定HASM和分化较差的HEK-293细胞中的AC相互作用蛋白。这 该项目提出了创新的多学科方法，以确定负责 建立和维持cAMP信号传导区室。我们的研究结果将具有广泛的适用性，因为 cAMP信号传导的基本性质，但也将与哮喘和COPD治疗直接相关。