Molecular signal transduction of cAMP compartments

cAMP 区室的分子信号转导

基本信息

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
项目类别：
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

项目摘要

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 (GPCR) 利用 cAMP 作为第二信使来改变细胞功能。事实上，在同一个细胞中，几种不同的 GPCR 可以增加 cAMP，这就产生了一个问题：细胞以不同的方式解释来自这些受体的信号。 cAMP 区室的概念，其中第二信使不是在整个细胞中均匀产生的，很容易被接受，但较差明白了。合成 cAMP 的酶，即腺苷酸环化酶 (AC)，并不是均匀分布在质膜。此外，GPCR 可以优先与某些 AC 亚型偶联，因为脂筏或非脂筏结构域中的共定位。虽然我们在了解具体情况方面取得了进展受体可以与不同的 AC 偶联，但在定义 cAMP 区室方面进展甚微细胞内部以及不同的 cAMP 池如何改变细胞反应。常用细胞模型是去分化的并且缺乏高度分化的 cAMP 池。然而，我们定义了两个清除原代人气道平滑肌 (HASM) 细胞中的 cAMP 信号区室。此举的目标该项目的目的是表征这两个 cAMP 区室中的关键监管成分、PDE 和 AKAP 并发现其中信号复合物的新蛋白质成员。我们将使用 siRNA 来敲除然后，各个 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化蛋白质组学定义了每个 cAMP 区室的磷酸化蛋白质组学特征，因此将利用这些签名来推断击倒后各个 PDE 或 AKAP 的角色。现状- 现有的光谱方法将直接评估细胞中cAMP的扩散。最后，我们将使用生物素接近度标记以识别 HASM 和分化较差的 HEK-293 细胞中的 AC 相互作用蛋白。这项目提出了创新的、多学科的方法来定义负责的组成部分建立和维持 cAMP 信号传导室。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，但也与哮喘和慢性阻塞性肺病治疗直接相关。