Molecular signal transduction of cAMP compartments

cAMP 区室的分子信号转导

基本信息

批准号：
10019564
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/10019564
关键词：
A kinase anchoring protein Adenylate Cyclase Adverse effects Asthma Biochemical Biological Models Biotin Buffers Cataloging Catalogs 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 kinase anchoring protein Adenylate Cyclase Adverse effects Asthma Biochemical Biological Models Biotin Buffers Cataloging Catalogs 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

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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.

大量的G蛋白耦合受体（GPCR）利用CAMP作为第二个信使来改变细胞功能。实际上，在同一细胞中，几种不同的GPCR可以增加营地，从而提出了一个问题。细胞从这些受体中解释信号。营地的概念，那里在整个牢房中，第二个信使不均匀地产生，但很容易接受但很差理解。合成营地，腺苷循环酶（AC）的酶不均匀分布质膜。此外，由于GPCR可以优先将其与某些AC同工型相结合脂质筏或非收割域中的共定位。尽管我们在理解如何具体的过程中取得了进展受体可以将其与不同的AC相结合，在定义营地的隔室方面几乎没有取得进展内部细胞以及如何通过不同的cAMP池修改细胞反应。常用细胞模型被脱离分化，缺乏高度分室的营地池。但是，我们定义了两个在原发性人类气道平滑肌（HASM）细胞中清除cAMP信号传导室。目标的目标项目是在这两个营地中表征关键的监管组件，PDE和AKAP 并发现其中信号复合物的新蛋白质成员。我们将用sirna敲除然后，单个PDE和AKAP通过新型荧光传感器测量局部cAMP信号。我们有使用定量磷蛋白质组学，定义了每个cAMP室的磷酸蛋白质组学特征，因此，将利用这些签名来推断敲低后单个PDE或AKAP的角色。最先进的艺术光谱法将直接评估CAMP在细胞中的扩散。最后，我们将使用生物素接近度标记以鉴定HASM和较不分化的HEK-293细胞中的交流相互作用蛋白。这项目提出了创新的多学科方法，以定义负责的组件建立和维护营地信号室。由于营地信号的基本性质，但也将与哮喘和COPD疗法有直接相关。