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隔间化提供了一个新的概念框架，以产生信号特异性和转导效率。尚未完全理解其建立，维护以及如何针对营地效应子的机制。使用甲状腺细胞，我们确定了一个新的亚膜室，其中ERM蛋白放射素支架将营地效应子EPAC和PKA带入三元络合物中。破坏这种隔室化的动作消除了TSH/CAMP介导的增殖，并成为开发新途径特异性抑制剂的基础。有趣的是，组成型活性RAP1的表达仅以其磷酸化形式（G12V-S179D）挽救了这种抑制作用，这表明该隔室的主要作用是将营地效应器定位在高cAMP浓度（即微域）的局部效应中，以最大程度地提高效应者的激活。证明了EPAC介导的激活的顺序顺序，然后证明了PKA介导的磷酸化。这设置了一个变构开关，其中PRAP1与其GEF分离，从而促进其与新的磷酸依赖性结合伴侣的关联，即CAP1（Cyclase-相关蛋白1）。我们的初步研究与PRAP1-CAP1一致，从而正调节cAMP合成的局部速率。我们在这里推进了积极反馈回路的假设，作为确保微域中当地cAMP水平的机制基础，以实现有效的效应子激活。因此，定义依赖PS179的变构开关所涉及的机制对于理解依赖磷酸化的RAP1-CAP控制CAMP动力学至关重要。我们将以两个集成的目标来实现这一目标。在AIM＃1中，NMR方法将被用来解决涉及磷酸化的变构沟通机制，以鉴定鉴定具有变构耦合的残基，州的种群及其交换动力学。在AIM＃2中，将使用生化和活细胞成像技术的组合来表征依赖磷酸的RAP1-CAP1相互作用及其积极调节分室cAMP合成的能力。该提案的长期目标是了解依赖CAMP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元的作用。了解负责CAMP隔室化的机制最终将为具有效应途径选择性的新特定抑制剂的合理设计提供见解。