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.