Mechanisms of Compartmentalized cAMP Signaling

标题：区室化cAMP信号传导机制 项目概要： 拟议研究的总体目标是提供一个关于如何循环的机械理解 AMP（cAMP）以精确的特异性调节多种不同的细胞功能。作为一个无处不在的第二 作为信使，cAMP调节对细胞生理学至关重要的多种过程，如细胞生长， 增殖、代谢、存活和移动性。在胰腺β细胞中，cAMP响应于葡萄糖而产生 或激素如胰高血糖素样肽-1（GLP-1），并反过来直接调节Ca 2+信号 影响胞吐胰岛素的释放以及调节基因表达，通过其效应子起作用 分子cAMP依赖性蛋白激酶（PKA）和由cAMP激活的交换蛋白（Epac）。的 “cAMP区室化”的概念，其中第二信使水平和效应子水平的独特变化 活动发生在空间和时间，提出了帮助理解精致的信号特异性， cAMP，但cAMP区室化的机制仍然难以捉摸。 在上一个融资期，我们有了突破性的发现。我们发现PKA 调节亚基RIα经历液-液相分离（LLPS），这些冷凝物是关键的 通过充当动态缓冲系统，并允许PDE产生cAMP， 微域这一发现是由于开发了一类新的荧光生物传感器， 可以靶向内源性水平的蛋白质，允许探测天然的信号动力学。 化学计量我们还创造了一类新型的荧光生物传感器，可以直接观察到 PKA的活动在超分辨率和阐明如何我们以前发现的振荡电路是受管制的 在空间上由A-激酶修饰蛋白79/150表达。我们总共发表了29篇同行评议的期刊文章 (with（4篇修订稿）。在目前的提案中，基于这些发现，我们将测试 我们假设PKA RIα相分离，通过螯合cAMP和PKA的催化亚基， 能够使cAMP信号传导区室化并使cAMP实现高信号传导特异性， β细胞的功能多样性。具体来说，我们将开发新的分子工具来询问 在活细胞中cAMP/PKA信号传导的时空调节，检查RIα相分离的影响 研究RI α对cAMP/PKA信号通路的影响，并确定RIα相分离在β细胞中的功能作用。 我们提出的研究将有助于更好地理解分子机制， cAMP/PKA信号传导的时空调节的功能作用，特别是在 调节细胞功能。由于cAMP信号通路中的畸变与临床状况有关， 如肥胖症和2型糖尿病，这样的理解应该有助于确定功能障碍的部位， 2型糖尿病和治疗干预的潜在目标。

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.