Molecular signal transduction of cAMP compartments

大量的G蛋白偶联受体利用cAMP作为第二信使来改变细胞 功能。事实上，在同一个细胞中，几种不同的gpr可以增加cAMP，这导致了一个问题： 细胞对来自这些受体的信号的解释是不同的。营地划分的概念，其中 第二信使并不是在整个细胞中均匀产生的，很容易被接受，但很难被接受 明白了。合成cAMP的酶，腺酰环化酶(Acs)，并不是均匀分布在 质膜。此外，由于以下原因，gpr可以优先偶联到某些AC亚型 共定位于脂筏或非RAFT结构域。虽然我们在理解具体情况方面取得了进展 受体可以与不同的ACS偶联，但在确定cAMP的区段方面进展甚微 在细胞内，以及不同cAMP池如何改变细胞反应。常用单元格 车型是去分化的，缺乏高度分隔的露营池。然而，我们定义了两个 人类原代呼吸道平滑肌(HASM)细胞中清晰的cAMP信号隔间。这样做的目的是 该项目的目的是确定这两个营区的关键管制组成部分--PDE和AKAP的特征 并发现其中的信号复合体的新的蛋白质成员。我们将使用siRNA来击倒 然后，单个PDE和AKAP通过新型荧光传感器测量局部cAMP信号。我们有 使用定量磷蛋白质组学定义每个cAMP隔室的磷蛋白质组特征， 因此将利用这些签名来推断击倒后单个PDE或AKAP的角色。最新情况- ART光谱方法将直接评估cAMP在细胞内的扩散。最后，我们将使用生物素近似性 标记以鉴定HASM和分化较差的HEK-293细胞中的AC相互作用蛋白。这 项目提出了创新的、多学科的方法来定义负责 建立和维护cAMP信号室。我们的发现将具有广泛的适用性，因为 CAMP信号的基本性质，但也将与哮喘和COPD的治疗直接相关。

A large number of G protein coupled receptors (GPCR) utilize cAMP as their second messenger to alter cell function. In fact, in the same cell several different GPCR can increase cAMP, leading to the question of how the cell interprets the signals from these receptors differently. The concept of cAMP compartmentation, where the second messenger is not generated uniformly throughout the cell, is readily accepted yet poorly understood. The enzymes that synthesize cAMP, adenylyl cyclases (ACs), are not uniformly distributed through the plasma membrane. Furthermore, GPCR can preferentially couple to certain AC isoforms due to colocalization in lipid rafts or non-raft domains. While we have made progress in understanding how specific receptors can couple to different ACs, little progress has been made in defining the compartments of cAMP inside cells and how cellular responses can be modified by different pools of cAMP. Commonly used cell models are de-differentiated and lack highly compartmentized cAMP pools. However, we have defined two clear cAMP signaling compartments in primary human airway smooth muscle (HASM) cells. The goal of this project is to characterize the key regulatory components, PDEs and AKAPs, in these two cAMP compartments and to discover novel protein members of signaling complexes therein. We will use siRNA to knockdown individual PDEs and AKAPs then measure localized cAMP signals via novel fluorescent sensors. We have defined the phosphoproteomic signatures of each cAMP compartment using quantitative phosphoproteomics, so will leverage these signatures to infer roles for individual PDEs or AKAPs following knockdown. State-of-the- art spectroscopic methods will directly assess the diffusion of cAMP in cells. Finally, we will use biotin proximity labeling to identify AC-interacting proteins in both HASM and less well differentiated HEK-293 cells. This project proposes innovative, multidisciplinary approaches to define the components responsible for establishing and maintaining cAMP signaling compartments. Our findings will have broad applicability due to the fundamental nature of cAMP signaling, but will also have direct relevance to asthma and COPD therapy.