Lisa M. DiPilato

Β2-Adrenergic Receptor Signaling and Desensitization Elucidated by Quantitative Modeling of Real Time cAMP Dynamics

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 β2-adrenergic receptor (β2AR) in living cells. β2AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of ∼10 μm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and β-arrestin to the regulation of β2AR 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 β-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of β2AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/β-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.

Imaging of cAMP Levels and Protein Kinase A Activity Reveals That Retinal Waves Drive Oscillations in Second-Messenger Cascades

Recent evidence demonstrates that low-frequency oscillations of intracellular calcium on timescales of seconds to minutes drive distinct aspects of neuronal development, but the mechanisms by which these calcium transients are coupled to signaling cascades are not well understood. Here we test the hypothesis that spontaneous electrical activity activates protein kinase A (PKA). We use live-cell indicators to observe spontaneous and evoked changes in cAMP levels and PKA activity in developing retinal neurons. Expression of cAMP and PKA indicators in neonatal rat retinal explants reveals spontaneous oscillations in PKA activity that are temporally correlated with spontaneous depolarizations associated with retinal waves. In response to short applications of forskolin, dopamine, or high-potassium concentration, we image an increase in cAMP levels and PKA activity, indicating that this second-messenger pathway can be activated quickly by neural activity. Depolarization-evoked increases in PKA activity were blocked by the removal of extracellular calcium, indicating that they are mediated by a calcium-dependent mechanism. These findings demonstrate for the first time that spontaneous activity in developing circuits is correlated with activation of the cAMP/PKA pathway and that PKA activity is turned on and off on the timescale of tens of seconds. These results show a link between neural activity and an intracellular biochemical cascade associated with plasticity, axon guidance, and neural differentiation.

Insulin Regulates Adipocyte Lipolysis via an Akt-Independent Signaling Pathway

ABSTRACT After a meal, insulin suppresses lipolysis through the activation of its downstream kinase, Akt, resulting in the inhibition of protein kinase A (PKA), the main positive effector of lipolysis. During insulin resistance, this process is ineffective, leading to a characteristic dyslipidemia and the worsening of impaired insulin action and obesity. Here, we describe a noncanonical Akt-independent, phosphoinositide-3 kinase (PI3K)-dependent pathway that regulates adipocyte lipolysis using restricted subcellular signaling. This pathway selectively alters the PKA phosphorylation of its major lipid droplet-associated substrate, perilipin. In contrast, the phosphorylation of another PKA substrate, hormone-sensitive lipase (HSL), remains Akt dependent. Furthermore, insulin regulates total PKA activity in an Akt-dependent manner. These findings indicate that localized changes in insulin action are responsible for the differential phosphorylation of PKA substrates. Thus, we identify a pathway by which insulin regulates lipolysis through the spatially compartmentalized modulation of PKA.

Regulation of nuclear PKA revealed by spatiotemporal manipulation of cyclic AMP

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.

The role of membrane microdomains in shaping β2-adrenergic receptor-mediated cAMP dynamics

Recently, membrane rafts and caveolae have received much attention for their role as signaling platforms, particularly due to their involvement in the pathogenesis of a number of diseases, including HIV as well as neurological and cardiovascular conditions. Signaling mediated by the beta-adrenergic receptor (beta-AR), a member of the large family of G-protein coupled receptors (GPCRs) that transduce extracellular messages via the ubiquitous second messenger, cAMP, has been a focus of raft studies since multiple components of the pathway are compartmentalized by these membrane microdomains. However, how these membrane rafts behave and regulate signaling dynamics in a cellular context is poorly understood. Here, we describe a live-cell assay based on single-cell, real-time fluorescence imaging, via an improved FRET-based cAMP biosensor, to monitor raft regulation of second messenger dynamics. Upon cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD), beta(2)-AR-mediated cAMP accumulation was enhanced and prolonged in HEK-293 cells, demonstrating that membrane raft integrity helps shape beta-AR signaling. Single-cell imaging in parallel with fractionation studies reveal that the enhancement and change of dynamics are mediated by the receptor and correlated with its redistribution. Finally, the effect of cholesterol depletion is receptor-type specific as MbetaCD treatment did not show the same effect when the raft-excluded prostaglandin E receptor was stimulated. This study highlights the potential of a live-cell, real-time imaging assay for studying membrane rafts, including high sensitivity and spatiotemporal resolution, to achieve a better understanding of the nuances of membrane microdomains in both healthy and diseased states.

The Role of PDE3B Phosphorylation in the Inhibition of Lipolysis by Insulin

ABSTRACT Inhibition of adipocyte lipolysis by insulin is important for whole-body energy homeostasis; its disruption has been implicated as contributing to the development of insulin resistance and type 2 diabetes mellitus. The main target of the antilipolytic action of insulin is believed to be phosphodiesterase 3B (PDE3B), whose phosphorylation by Akt leads to accelerated degradation of the prolipolytic second messenger cyclic AMP (cAMP). To test this hypothesis genetically, brown adipocytes lacking PDE3B were examined for their regulation of lipolysis. In Pde3b knockout (KO) adipocytes, insulin was unable to suppress β-adrenergic receptor-stimulated glycerol release. Reexpressing wild-type PDE3B in KO adipocytes fully rescued the action of insulin against lipolysis. Surprisingly, a mutant form of PDE3B that ablates the major Akt phosphorylation site, murine S273, also restored the ability of insulin to suppress lipolysis. Taken together, these data suggest that phosphorylation of PDE3B by Akt is not required for insulin to suppress adipocyte lipolysis.

Fluorescent protein-based biosensors: resolving spatiotemporal dynamics of signaling

Cellular processes are orchestrated by the precise coordination and regulation of molecular events in the cell. Fluorescent protein-based biosensors coupled with live-cell imaging have enabled the visualization of these events in real time and helped shape some of the current concepts of signal transduction, such as spatial compartmentation. The quantitative information produced by these tools has been incorporated into mathematical models that are capable of predicting highly complex and dynamic behaviors of cellular signaling networks, thus providing a systems level understanding of how pathways interact to produce a functional response. Finally, with technological advances in high-throughput and in vivo imaging, these molecular tools promise to continually engender significant contributions to our understanding of cellular processes under normal and diseased conditions.

A long lasting β1 adrenergic receptor stimulation of cAMP/protein kinase A (PKA) signal in cardiac myocytes.

Background: Transient β1AR activation remains at odds with long lasting cellular and physiological responses. Results: The agonist-occupied β1AR continuously signals to adenylyl cyclase (AC) to produce cAMP in both cardiac myocytes and neurons for more than 8 h, which is masked by receptor-associated PDE4D8. Conclusion: Stimulation of β1AR induces long-lasting cAMP production in the heart for ligand-induced physiological responses. Significance: We show a novel mechanism to understand persistent β1AR signaling in the heart. Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β1 adrenergic receptor (β1AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t½ of 3.9 h) in cardiac myocytes. However, the β1AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β1AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β1AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response.

Dynamic Visualization of Signaling Activities in Living Cells

Engineered fluorescent reporters allow researchers to follow subcellular activities of signaling components in real time in live cells. The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation and dynamic modulation of the activities of protein kinases, phosphatases, and second messengers. However, traditional methodologies for detecting signaling events, such as activation of kinases and second-messenger production and degradation, are limited in their spatiotemporal resolution and do not allow one to follow these events within the live-cell context. To achieve dynamic tracking of signaling activities in living cells, we have engineered genetically encoded fluorescent reporters for protein kinases and second messengers, such as cyclic adenosine monophosphate (cAMP) and phosphoinositides. Their development and specific examples of their application are discussed. In addition, a live-cell, high-throughput screening method has been developed for identification of new modulators that affect the dynamic activity of kinases and second messengers. Together, these reporters have the potential to provide important spatiotemporal information about the circuitry governing specific signaling events in living cells.

The role of mouse Akt2 in insulin-dependent suppression of adipocyte lipolysis in vivo

Aim/hypothesisThe release of fatty acids from adipocytes, i.e. lipolysis, is maintained under tight control, primarily by the opposing actions of catecholamines and insulin. A widely accepted model is that insulin antagonises catecholamine-dependent lipolysis through phosphorylation and activation of cAMP phosphodiesterase 3B (PDE3B) by the serine-threonine protein kinase Akt (protein kinase B). Recently, this hypothesis has been challenged, as in cultured adipocytes insulin appears, under some conditions, to suppress lipolysis independently of Akt.MethodsTo address the requirement for Akt2, the predominant isoform expressed in classic insulin target tissues, in the suppression of fatty acid release in vivo, we assessed lipolysis in mice lacking Akt2.ResultsIn the fed state and following an oral glucose challenge, Akt2 null mice were glucose intolerant and hyperinsulinaemic, but nonetheless exhibited normal serum NEFA and glycerol levels, suggestive of normal suppression of lipolysis. Furthermore, insulin partially inhibited lipolysis in Akt2 null mice during an insulin tolerance test (ITT) and hyperinsulinaemic–euglycaemic clamp, respectively. In support of these in vivo observations, insulin antagonised catecholamine-induced lipolysis in primary brown fat adipocytes from Akt2-deficient mice.Conclusions/interpretationThese data suggest that suppression of lipolysis by insulin in hyperinsulinaemic states can take place in the absence of Akt2.