Spatiotemporal visualization of adenylyl cyclase signaling

项目摘要/摘要 环磷酸腺苷(CAMP)是调节多种细胞信号通路的重要二级信使 贯穿整个生理学。腺酰环化酶(AC)在G蛋白的作用下催化ATP转化为cAMP。 偶联受体信号传递，是细胞信号传递的重要中继站和整合中心。 目前，急性冠脉综合征是治疗一系列疾病的未开发的靶点，包括癌症、慢性疾病 阻塞性肺病、神经精神障碍、糖尿病、心脏病和药物滥用。在 九个基因的细胞背景、差异表达、区划和微域定位 跨膜AC异构体引起一系列可能的信号转导结果，使我们的 治疗靶向这一细胞信号的关键变阻器的能力。我们对交流的机械理解 功能有限，只有最近第一次瞥见冷冻电子揭示的全长AC-GαS结构 显微镜(低温电子显微镜)。然而，隔离状态下的交流的静态快照形成了不完整的图像 在活体内发生的时空信号。交流信号的分子和机制细节的缺乏 原生环境中的程序集在我们的理解中留下了空白，进一步制约了发展 针对AC和营地通路的药物。为了解决这一差距，这一计划的首要目标是 建议捕获腺酰环化酶和信号伙伴之间生理上相关的复合体 在近乎本地的和蜂窝环境中。这将通过以下具体目标来实现： 腺酰环化酶功能信号组件的结构和生化特征，以及(2) 腺酰环化酶微结构域形态及其在环化酶激活后的动态变化 SITE。完成这些目标将代表着腺酰环化酶生物学的重大飞跃，提供 为进一步研究腺酰环化酶奠定了基础。这项工作建立在我的G蛋白生物化学基础上 细胞信号分析、蛋白质纯化和荧光显微镜方面的背景和经验。 利用我的导师李博士的实验室提供的世界级培训和资源。 Georgios Skiniotis，拟议的研究还提供了获得结构方面的专业知识的机会 生物学，低温电子断层扫描，蛋白质组学方法，提高了我作为研究人员的能力，以及 发展成为腺酰环化酶领域的新兴领导者。因此，我将处于有利地位，能够建立 一项成功资助独立研究的计划。

PROJECT SUMMARY/ABSTRACT Cyclic AMP (cAMP) is a critical secondary messenger that modulates many cell signaling pathways throughout physiology. Adenylyl cyclase (AC) catalytically converts ATP to cAMP in response to G protein- coupled receptor signaling, thus acting as an essential relay and integration center for cell signaling. Currently, ACs represent an unexploited target for treating a range of diseases including cancer, chronic obstructive pulmonary disease, neuropsychiatric disorders, diabetes, heart disease, and drug abuse. Within a cellular context, differential expression, compartmentalization, and microdomain localization of nine transmembrane AC isoforms give rise to a broad range of possible signaling outcomes, complicating our ability to therapeutically target this critical rheostat of cell signaling. Our mechanistic understanding of AC function is limited, with only a recent first glimpse of a full-length AC-Gαs structure revealed by cryo-electron microscopy (cryoEM). However, the static snapshot of AC in isolation forms an incomplete picture of the spatiotemporal signaling that occurs in vivo. The lack of molecular and mechanistic details of AC signaling assemblies in native environments leaves a gap in our understanding, further restricting the development of pharmaceuticals targeting AC and cAMP pathways. To address this gap, the overarching goal of this proposal is to capture physiologically relevant complexes between adenylyl cyclase and signaling partners in near-native and cellular environments. This will be achieved through the following specific aims: (1) structurally and biochemically characterize a functional signaling assembly of adenylyl cyclase, and (2) visualize adenylyl cyclase microdomain topography and its dynamics in response to cyclase activation in situ. Completing these aims will represent a substantial leap forward in adenylyl cyclase biology, providing the framework for further adenylyl cyclase study. This work builds upon my G protein biochemistry background and experience with cell signaling assays, protein purification, and fluorescence microscopy. Taking advantage of the world-class training and resources available in the laboratory of my mentor, Dr. Georgios Skiniotis, the proposed studies also provide an opportunity to acquire expertise in structural biology, cryo-electron tomography, proteomic approaches, sharpen my abilities as a researcher, and develop as an emerging leader in the adenylyl cyclase field. As a result, I will be well-positioned to establish a program of successfully funded independent research.