cAMP effector pathways in TSH signaling

描述（由申请人提供）：cAMP区隔化为产生信号特异性和转导效率提供了一个新的概念框架。其建立和维持的机制以及cAMP效应物如何靶向其尚不完全清楚。利用甲状腺细胞，我们发现了一个新的亚膜腔室，在那里ERM蛋白放射素将cAMP效应物Epac和PKA支架成一个三元复合物。破坏这种区隔化的操作消除了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，组成活性Rap1的表达，但仅在其磷酸化形式（G12V-S179D）中才能恢复这种抑制，这表明该区室的主要作用是将cAMP效应物定位在高cAMP浓度的局部区域（即微域），以最大限度地激活效应物。epac介导的激活和pka介导的磷酸化顺序被证实。这设置了一个变构开关，其中pRap1从其GEF中解离，促进其与新的磷酸化依赖结合伙伴CAP1（环化酶相关蛋白1）的结合。我们的初步研究结果与pRap1-CAP1正调节cAMP的局部合成速率一致。我们在这里提出了一个正反馈回路的假设，作为保证微域中局部cAMP水平为有效的效应激活而优化的机制基础。因此，确定ps179依赖性变弹性开关的相关机制对于理解磷酸化依赖性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来实现这一目标。在目标#1中，将利用核磁共振方法来解决涉及磷酸依赖的变构通信的机制，旨在识别变构耦合的残基，状态的人口及其交换动力学。在Aim #2中，生化和活细胞成像技术的结合将用于表征磷酸化依赖性Rap1-CAP1相互作用及其积极调节区隔化cAMP合成的能力。本研究的长期目标是了解camp依赖性信号事件的时空调控，以及Rap1及其磷酸化状态作为信号整合单元的作用。了解cAMP区隔化的机制将最终为合理设计具有效应通路选择性的新型特异性抑制剂提供见解。

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.