FRET ͉ cAMP-dependent protein kinase

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous ␤ 2 -adrenergic receptor (␤ 2 AR) in living cells. ␤ 2 AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of ϳ10 M and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and ␤-arrestin to the regulation of ␤ 2 AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to ␤-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of ␤ 2 AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMPdependent kinase is insignificant but that GRK6/␤-arrestin-mediated inactivation is rapid and profound, occurring with a halftime of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.Tachyphylaxis, or desensitization, denoting the attenuation of a biological response to sustained or repeated intervention, is a pervasive phenomenon in physiological systems. For G protein-coupled receptors (GPCRs) 7 (or, more broadly, seventransmembrane receptors), desensitization occurs through molecular mechanisms that can profoundly limit further stimulation of downstream signals, either through direct receptor inactivation or inhibition of downstream signaling. At the physiological level, we refer to this general loss of responsiveness as desensitization; we refer to the more specific case of direct inhibition of receptor molecules as "receptor inactivation." At the level of the receptor, GPCR signals represent a dynamic balance between ligand-stimulated activities, such as G protein coupling, and negative feedback mechanisms, such as receptor phosphorylation and ␤-arrestin recruitment (1). An agonist's efficacy is determined by the balance between these activities and is limited by the kinetics of receptor inactivation. Despite intensive research into the molecular mechanisms of desensitization by GPCR inactivation, the relative contributions of these mechanisms are largely unknown. One reason for this is that, until recently, there have been few techniques that directly measure receptor signaling in real time. Rather, data showing desensitization and receptor inactivation are usually based on either physiological assessments, such as hemodynamic parameters, whi...

Understanding how specific cAMP signals are organized and relayed to their effectors in different compartments of the cell to achieve functional specificity requires molecular tools that allow precise manipulation of cAMP in these compartments. Here we characterize a new method using bicarbonate-activatable and genetically targetable soluble adenylyl cyclase (sAC) to control the location, kinetics and magnitude of the cAMP signal. Using this live-cell cAMP manipulation in conjunction with fluorescence imaging and mechanistic modeling, we uncover the activation of a resident pool of PKA holoenzyme in the nuclei of HEK-293 cells, modifying the existing dogma of cAMP-PKA signaling in the nucleus. Furthermore, we show that phosphodiesterases (PDE) and A-Kinase Anchoring Proteins (AKAP) are critical in shaping nuclear PKA responses. Collectively, our data suggests a new model where AKAP-localized PDEs tune an activation threshold for nuclear PKA holoenzyme, thereby converting spatially distinct second messenger signals to temporally controlled nuclear kinase activity.