cAMP effector pathways in TSH signaling

TSH 信号转导中的 cAMP 效应通路

• 批准号: 8501799
• 负责人: DANIEL L ALTSCHULER
• 金额: $ 37.14万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8501799
• 关键词: Address; Area; Binding; Biochemical; Biological; Biological Process; Cell Proliferation; Cells; Communication; Complex; Coupled; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Development; Disease; ERM protein; Endocrine system; Event; Feedback; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Hormones; Human; Imaging Techniques; Life; Link; Maintenance; Mediating; Membrane; Monomeric GTP-Binding Proteins; Mutation; Normal Cell; Outcome; Pathway interactions; Phosphorylation; Physiology; Population; Positioning Attribute; Proteins; Recruitment Activity; Regulation; Role; Second Messenger Systems; Signal Transduction; Solutions; Specificity; Structure; Testing; Thyroid Gland; base; cell type; cellular imaging; design; inhibitor/antagonist; insight; public health relevance; radixin protein; scaffold; second messenger; transduction efficiency; tumorigenesis

DESCRIPTION (provided by applicant): cAMP compartmentalization provides a new conceptual framework to generate signaling specificity and transduction efficiency. The mechanisms involved in its establishment, maintenance and how cAMP effectors are targeted to it are not completely understood. Using thyroid cells we identified a new sub-membrane compartment, where the ERM protein radixin scaffolds both cAMP effectors Epac and PKA into a ternary complex. Maneuvers that disrupt this compartmentalization abrogate TSH/cAMP-mediated proliferation, and served as the basis for the development of new pathway-specific inhibitors. Interestingly, expression of constitutively active Rap1 but only in its phosphorylated form (G12V-S179D) rescues this inhibition, indicating the main role of this compartment is to position cAMP effectors in a local area of high cAMP concentration (i.e. a microdomain) to maximize effector activation. A sequential order of Epac-mediated activation followed by PKA-mediated phosphorylation was demonstrated. This sets an allosteric switch where pRap1 dissociates from its GEF promoting its association with new phospho-dependent binding partners, i.e. CAP1 (Cyclase- Associated Protein 1). Our preliminary studies are consistent with pRap1-CAP1 positively modulating the localized rate of cAMP synthesis. We advance here the hypothesis of a positive feedback loop as the mechanistic basis assuring that local cAMP levels in the microdomain are optimized for efficient effector activation. Defining the mechanisms involved in the pS179-dependent allosteric switch is therefore critical for the understanding of the phospho-dependent Rap1-CAP control of cAMP dynamics. We will accomplish this in two integrated aims. In Aim #1 NMR approaches will be utilized to address the mechanism involved in the phospho-dependent allosteric communication aiming at the identification of the residues that are allosterically coupled, the population of the states and their exchange dynamics. In Aim #2 a combination of biochemical and live cell imaging techniques will be used to characterize the phospho-dependent Rap1-CAP1 interaction and its ability to positively modulate compartmentalized cAMP synthesis. The long-term goal of this proposal is to understand the spatial and temporal regulation of the cAMP-dependent signaling events, and the role of Rap1 and its phosphorylation state as a signal integration unit. Understanding the mechanisms responsible for cAMP compartmentalization will eventually provide insights into the rational design of new specific inhibitors with effector pathway selectivity.

描述（由申请人提供）：cAMP区室化提供了产生信号传导特异性和转导效率的新概念框架。其建立、维持和cAMP效应物如何靶向其的机制尚未完全了解。使用甲状腺细胞，我们确定了一个新的亚膜室，其中ERM蛋白radixin支架cAMP效应子Epac和PKA成为三元复合物。破坏这种区室化的策略消除了TSH/cAMP介导的增殖，并作为开发新的途径特异性抑制剂的基础。有趣的是，组成型活性Rap 1的表达但仅以其磷酸化形式（G12 V-S179 D）挽救了这种抑制，表明该隔室的主要作用是将cAMP效应物定位在高cAMP浓度的局部区域（即微结构域）以使效应物活化最大化。Epac介导的活化随后是PKA介导的磷酸化的顺序被证明。这设置了变构开关，其中pRap 1从其GEF解离，促进其与新的磷酸依赖性结合配偶体，即CAP 1（环化酶相关蛋白1）的结合。我们的初步研究与pRap 1-CAP 1正调节cAMP合成的局部速率一致。在这里，我们提出了一个正反馈回路的假设，作为确保局部cAMP水平在微域有效的效应激活优化的机制基础。因此，定义参与pS179依赖性变构开关的机制对于理解cAMP动力学的磷酸依赖性Rap 1-CAP控制至关重要。我们将通过两个综合目标来实现这一目标。在目标#1中，NMR方法将用于解决磷酸依赖性变构通信中涉及的机制，旨在鉴定变构偶联的残基、状态的群体及其交换动力学。在目标#2中，生物化学和活细胞成像技术的组合将用于表征磷酸依赖性Rap 1-CAP 1相互作用及其正调节区室化cAMP合成的能力。该提案的长期目标是了解cAMP依赖性信号事件的空间和时间调节，以及Rap 1及其磷酸化状态作为信号整合单元的作用。了解负责cAMP区室化的机制将最终提供具有效应通路选择性的新的特异性抑制剂的合理设计的见解。