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划分提供了一个新的概念框架来产生信号特异性和转导效率。它的建立、维持所涉及的机制以及营地效应者是如何针对它的还没有完全了解。利用甲状腺细胞，我们确定了一个新的亚膜室，在那里ERM蛋白Radisin将cAMP效应器EPAC和PKA构建成一个三元复合体。破坏这种区划的动作可以阻断TSH/cAMP介导的增殖，并为开发新的通路特异性抑制剂奠定了基础。有趣的是，仅以其磷酸化形式表达具有结构性活性的Rap1(G12V-S179D)可以挽救这种抑制，表明这个隔室的主要作用是将cAMP效应器定位在cAMP浓度较高的局部区域(即微区)，以最大化效应器的激活。EPAC介导的激活和PKA介导的磷酸化是连续的顺序。这设置了一个变构开关，其中pRap1从其环境基金解离，促进其与新的磷酸依赖结合伙伴，即CAP1(环酶相关蛋白1)的结合。我们的初步研究与pRap1-cap1正向调节cAMP合成的定位速率是一致的。我们在这里提出了一个正反馈环的假设作为机制基础，确保微域中的局部cAMP水平为有效的效应器激活而优化。因此，确定pS179依赖的变构开关所涉及的机制对于理解磷酸依赖的Rap1-CAP对cAMP动力学的控制至关重要。我们将通过两个综合目标实现这一目标。在AIM#1中，将利用核磁共振方法来研究磷依赖的变构通信所涉及的机制，目的是识别变构偶联的残基、态的布居及其交换动力学。在AIM#2中，将使用生物化学和活细胞成像技术相结合的方法来表征依赖于磷酸的Rap1-Cap1相互作用及其积极调节区段cAMP合成的能力。这一建议的长期目标是了解cAMP依赖的信号事件的空间和时间调节，以及Rap1及其磷酸化状态作为信号整合单位的作用。了解cAMP区划的机制最终将为合理设计具有效应通路选择性的新的特异性抑制剂提供见解。