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Spatiotemporal Regulation of PKA in Response to beta-Adrenergic Stimulation

PKA 对 β-肾上腺素能刺激的时空调节

基本信息

批准号：
8010190
负责人：
Charlene Depry
金额：
$ 4.18万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-16 至 2012-01-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8010190
关键词：
A kinase anchoring protein Action Potentials Adrenergic Agents Adrenergic Receptor Affect Agonist American Apoptosis Cardiac Cardiac Myocytes Cardiac Output Caveolae Cell Differentiation process Cell membrane Cell physiology Cells Cessation of life Cholesterol Cyclic AMP-Dependent Protein Kinases Development Diagnosis Diffuse Embryo Event Fluorescence Resonance Energy Transfer Genetic Transcription Goals Heart Hypertrophy Heart Rate Heart failure Human Cell Line Individual Ion Channel Kidney Kinetics Knowledge Life Link Location Measurement Measures Membrane Microdomains Metabolism Monitor Muscle Peptides Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Play Protein Kinase Proteins Protocols documentation Receptor Activation Regulation Relaxation Reporter Research Research Activity Resolution Role Scotland Signal Transduction Specificity Structure Techniques Therapeutic Time Validation adrenergic base cell growth heart function improved phosphoric diester hydrolase receptor receptor-mediated signaling research study response spatiotemporal therapy development tool

项目摘要

DESCRIPTION (provided by applicant): The overall goal of this research is to elucidate the mechanisms involved in regulating cAMP dependent protein kinase (PKA) activity that give rise to high specificity signaling in response to Padrenergic receptor (P-AR) stimulation. Given the broad spectrum of signaling events that are controlled by this kinase, it is crucial that proper phosphorylation by PKA occur at the right time in the right location within a cell, in order to attain selective and specific cellular responses. As such, deregulation of PKA in cardiomyocytes is often associated with heart failure. The tools needed to study live-cell PKA activity have recently become available and will allow for effective exploration of these mechanisms. Specific aim 1 of this project will compare P-AR stimulated spatiotemporal dynamics of PKA activity by stimulating and/or inhibiting p-ARs with receptor subtype specific agonists and antagonists. Specific aim 2 will attempt to determine the roles that membrane microdomains and spatially compartmentalized p-ARs play in the regulation of PKA dynamics by disrupting membrane microdomain structure (via cholesterol depletion) and monitoring local PKA activity. Specific aim 3 will attempt to investigate the roles that A-kinase anchoring proteins (AKAPs) play in regulating P-AR stimulated PKA dynamics by disrupting AKAPs and then monitoring PKA activity at subcellular locations. Each of these aims is to be carried out in cardiomyocytes in an attempt to fill in the current gaps in our knowledge about mechanisms underlying PKA regulation that links receptor activation to specific cardiac output (ie. heart rate, contraction force, relaxation). Additionally, each aim requires live-cell measurement of PKA activity, which will utilize a FRET-based probe for spatiotemporal resolution of PKA activity. This research should provide more in-depth understanding about basic cardiomyocyte signaling mechanisms and aid in the development of treatment and diagnosis of heart failure.

描述（由申请方提供）：本研究的总体目标是阐明调节cAMP依赖性蛋白激酶（PKA）活性的机制，该活性在对β-肾上腺素能受体（P-AR）刺激的反应中产生高特异性信号传导。鉴于由这种激酶控制的广泛的信号事件，PKA的适当磷酸化在细胞内的正确位置在正确的时间发生是至关重要的，以获得选择性和特异性的细胞反应。因此，心肌细胞中PKA的失调通常与心力衰竭有关。研究活细胞PKA活性所需的工具最近已经可用，并将允许有效探索这些机制。本项目的具体目标1将通过用受体亚型特异性激动剂和拮抗剂刺激和/或抑制p-AR来比较p-AR刺激PKA活性的时空动力学。具体目标2将试图通过破坏膜微结构域结构（通过胆固醇消耗）和监测局部PKA活性来确定膜微结构域和空间区室化的p-AR在PKA动力学调节中的作用。具体目标3将试图通过破坏AKAP然后监测亚细胞位置的PKA活性来研究A-激酶锚定蛋白（AKAP）在调节P-AR刺激的PKA动力学中发挥的作用。这些目标中的每一个都是在心肌细胞中进行的，试图填补我们目前关于PKA调节机制的知识空白，PKA调节机制将受体激活与特定的心输出量（即，心率、收缩力、松弛）。此外，每个目标都需要活细胞测量PKA活性，这将利用基于FRET的探针对PKA活性进行时空分辨率。这项研究应该提供对基本心肌细胞信号传导机制的更深入理解，并有助于心力衰竭治疗和诊断的发展。