Mechanisms of Compartmentalized cAMP Signaling

CAMP信号的分区化机制 项目总结： 拟议研究的总体目标是提供对如何循环的机械性理解 AMP(CAMP)以精致的特异性调节多种不同的细胞功能。作为无处不在的第二个 CAMP调节多种对细胞生理至关重要的过程，如细胞生长、 增殖、新陈代谢、生存和活动能力。在胰腺β细胞中，cAMP是对葡萄糖的反应而产生的。 或激素，如胰高血糖素样肽-1(GLP-1)，进而直接调节钙信号 通过其效应器影响胞吐胰岛素的释放并调节基因的表达 CAMP依赖的蛋白激酶(PKA)和cAMP激活的交换蛋白(EPAC)分子。这个 “cAMP分区”的概念，其中第二信使水平和效应器的独特变化 活动既发生在空间上，又发生在时间上，被提出有助于理解精细的信号特异性 然而，营地划分的基本机制仍然难以捉摸。 在之前的资助期，我们有了一个突破性的发现。我们发现PKA 调节亚基RIα经历液-液相分离(LLP)，这些凝析油是关键的 通过充当动态缓冲系统进行营地划分，并允许PDE创建营地 微域。这一发现是由一种新型荧光生物传感器的开发实现的 可以在内源水平上针对蛋白质，允许在天然水平上探测信号动力学 化学计量学。我们还创造了一种新型的荧光生物传感器，可以直接可视化 超分辨中的PKA活性，并阐明了我们先前发现的振荡电路是如何调节的 空间上由A-激酶锚定蛋白79/150决定。我们总共发表了29篇同行评议的期刊文章 (另有4篇手稿正在修订中)在目前的提案中，在这些发现的基础上，我们将测试 我们的假设是，PKA RIα相分离，通过隔离cAMP和PKA的催化亚单位， 启用划分的cAMP信号，并允许cAMP实现高信号特异性和 β细胞的功能多样性。具体地说，我们将开发新的分子工具来审问 活细胞内cAMP/PKA信号的时空调节，检测RIα时相分离的影响 对cAMP/PKA信号的影响，并确定RIα时相分离在β细胞中的作用。 我们建议的研究应该有助于更好地理解分子机制和 CAMP/PKA信号的时空调控的功能作用，特别是在 细胞功能的调节。CAMP信号通路中的AS异常与临床情况有关 例如肥胖和2型糖尿病，这样的理解应该有助于识别 2型糖尿病和治疗干预的潜在靶点。

Title: Mechanisms of Compartmentalized cAMP Signaling Project summary: The overall goal of the proposed research is to provide a mechanistic understanding about how cyclic AMP (cAMP) regulates a variety of different cellular functions with exquisite specificity. As a ubiquitous second messenger, cAMP regulates a variety of processes that are critical to cell physiology, such as cell growth, proliferation, metabolism, survival and mobility. In pancreatic β cells, cAMP is produced in response to glucose or hormones such as glucagon-like peptide-1 (GLP-1), and in turn modulates the Ca2+ signal, directly influencing exocytotic insulin release as well as regulating gene expression, acting through its effector molecules cAMP-dependent protein kinase (PKA) and exchange proteins activated by cAMP (Epac). The concept of “cAMP compartmentation”, wherein unique changes in second messenger levels and effector activities occur in both space and time, was put forward to help understand the exquisite signaling specificity of cAMP, yet the mechanisms underlying cAMP compartmentation remain elusive. In the previous funding period, we made a breakthrough discovery. We discovered that the PKA regulatory subunit RIα undergoes liquid-liquid phase separation (LLPS) and that these condensates are critical for cAMP compartmentation by serving as a dynamic buffering system, and allowing PDEs to create cAMP microdomains. This discovery was enabled by the development of a new class of fluorescent biosensors that can be targeted to proteins at the endogenous level, allowing probing of signaling dynamics at the native stoichiometry. We have also created a novel class of fluorescent biosensors that allow direct visualization of PKA activities in superresolution and elucidated how our previously discovered oscillatory circuit is regulated spatially by A-Kinase Anchoring Protein 79/150. Altogether we published 29 peer-reviewed journal articles (with 4 additional manuscripts in revision). In the current proposal, building on these discoveries, we will test our hypothesis that PKA RIα phase separation, by sequestering cAMP and the catalytic subunit of PKA, enables compartmentalized cAMP signaling and allows cAMP to achieve high signaling specificity and functional diversity in β cells. Specifically, we will develop novel molecular tools to interrogate the spatiotemporal regulation of cAMP/PKA signaling in living cells, examine the impact of RIα phase separation on cAMP/PKA signaling and determine the functional roles of RIα phase separation in β cells. Our proposed studies should lead to a better understanding of the molecular mechanisms and functional roles of the spatiotemporal regulation of cAMP/PKA signaling, particularly in the context of the regulation of  cell functions. As aberrations in the cAMP signaling pathway are implicated in clinical conditions such as obesity and type 2 diabetes mellitus, such an understanding should help identify sites of dysfunction in type 2 diabetes and potential targets for therapeutic intervention.