Phosphodiesterase in heart and vessels: from physiology to diseases.

Phosphodiesterases (PDEs) are a superfamily of enzymes that hydrolyze cyclic nucleotides, including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both cyclic nucleotides are critical secondary messengers in the neurohormonal regulation in the cardiovascular system. PDEs precisely control spatiotemporal subcellular distribution of cyclic nucleotides in a cell- and tissue-specific manner, playing critical roles in physiological responses to hormone stimulation in the heart and vessels. Dysregulation of PDEs has been linked to the development of several cardiovascular diseases, such as hypertension, aneurysm, atherosclerosis, arrhythmia, and heart failure. Targeting these enzymes has been proven effective in treating cardiovascular diseases and is an attractive and promising strategy for the development of new drugs. In this review, we discuss the current understanding of the complex regulation of PDE isoforms in cardiovascular function, highlighting the divergent and even opposing roles of PDE isoforms in different pathogenesis.

Elucidation of a dynamic interplay between a beta-2 adrenergic receptor, its agonist, and stimulatory G protein.

G protein-coupled receptors (GPCRs) represent the largest group of membrane receptors for transmembrane signal transduction. Ligand-induced activation of GPCRs triggers G protein activation followed by various signaling cascades. Understanding the structural and energetic determinants of ligand binding to GPCRs and GPCRs to G proteins is crucial to the design of pharmacological treatments targeting specific conformations of these proteins to precisely control their signaling properties. In this study, we focused on interactions of a prototypical GPCR, beta-2 adrenergic receptor (ß2AR), with its endogenous agonist, norepinephrine (NE), and the stimulatory G protein (Gs). Using molecular dynamics (MD) simulations, we demonstrated the stabilization of cationic NE, NE(+), binding to ß2AR by Gs protein recruitment, in line with experimental observations. We also captured the partial dissociation of the ligand from ß2AR and the conformational interconversions of Gs between closed and open conformations in the NE(+)-ß2AR-Gs ternary complex while it is still bound to the receptor. The variation of NE(+) binding poses was found to alter Gs α subunit (Gsα) conformational transitions. Our simulations showed that the interdomain movement and the stacking of Gsα α1 and α5 helices are significant for increasing the distance between the Gsα and ß2AR, which may indicate a partial dissociation of Gsα The distance increase commences when Gsα is predominantly in an open state and can be triggered by the intracellular loop 3 (ICL3) of ß2AR interacting with Gsα, causing conformational changes of the α5 helix. Our results help explain molecular mechanisms of ligand and GPCR-mediated modulation of G protein activation.

Disruption of mitochondria-sarcoplasmic reticulum microdomain connectomics contributes to sinus node dysfunction in heart failure.

The sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not completely understood. Mitochondria are critical to cellular processes that determine the life or death of the cell. The release of Ca2+ from the ryanodine receptors 2 (RyR2) on the sarcoplasmic reticulum (SR) at mitochondria-SR microdomains serves as the critical communication to match energy production to meet metabolic demands. Therefore, we tested the hypothesis that alterations in the mitochondria-SR connectomics contribute to SAN dysfunction in HF. We took advantage of a mouse model of chronic pressure overload-induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR-Cas9-mediated knockdown of mitofusin-2 (Mfn2), the mitochondria-SR tethering GTPase protein. TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased mitochondria-SR distance. The expression of Mfn2 was significantly down-regulated and showed reduced colocalization with RyR2 in HF SAN cells. Indeed, SAN-specific Mfn2 knockdown led to alterations in the mitochondria-SR microdomains and SAN dysfunction. Finally, disruptions in the mitochondria-SR microdomains resulted in abnormal mitochondrial Ca2+ handling, alterations in localized protein kinase A (PKA) activity, and impaired mitochondrial function in HF SAN cells. The current study provides insights into the role of mitochondria-SR microdomains in SAN automaticity and possible therapeutic targets for SAN dysfunction in HF patients.

Intracellular ß1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility.

RATIONALE: ß1ARs (ß1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular ß1AR in cardiac contractility remains to be elucidated. OBJECTIVE: Test localization and function of intracellular ß1AR on cardiac contractility. METHODS AND RESULTS: Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of ß1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant ßAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant ßAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility. CONCLUSIONS: Functional ß1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular ß1ARs requires catecholamine transport via OCT3.

Monoamine oxidase A and organic cation transporter 3 coordinate intracellular ß1AR signaling to calibrate cardiac contractile function.

We have recently identified a pool of intracellular ß1 adrenergic receptors (ß1ARs) at the sarcoplasmic reticulum (SR) crucial for cardiac function. Here, we aim to characterize the integrative control of intracellular catecholamine for subcellular ß1AR signaling and cardiac function. Using anchored Förster resonance energy transfer (FRET) biosensors and transgenic mice, we determined the regulation of compartmentalized ß1AR-PKA signaling at the SR and plasma membrane (PM) microdomains by organic cation transporter 3 (OCT3) and monoamine oxidase A (MAO-A), two critical modulators of catecholamine uptake and homeostasis. Additionally, we examined local PKA substrate phosphorylation and excitation-contraction coupling in cardiomyocyte. Cardiac-specific deletion of MAO-A (MAO-A-CKO) elevates catecholamines and cAMP levels in the myocardium, baseline cardiac function, and adrenergic responses. Both MAO-A deletion and inhibitor (MAOi) selectively enhance the local ß1AR-PKA activity at the SR but not PM, and augment phosphorylation of phospholamban, Ca2+ cycling, and myocyte contractile response. Overexpression of MAO-A suppresses the SR-ß1AR-PKA activity and PKA phosphorylation. However, deletion or inhibition of OCT3 by corticosterone prevents the effects induced by MAOi and MAO-A deletion in cardiomyocytes. Deletion or inhibition of OCT3 also negates the effects of MAOi and MAO-A deficiency in cardiac function and adrenergic responses in vivo. Our data show that MAO-A and OCT3 act in concert to fine-tune the intracellular SR-ß1AR-PKA signaling and cardiac fight-or-flight response. We reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of these drugs with cardiac implications.

Probing spatiotemporal PKA activity at the ryanodine receptor and SERCA2a nanodomains in cardomyocytes.

Spatiotemporal regulation of subcellular protein kinase A (PKA) activity for precise substrate phosphorylation is essential for cellular responses to hormonal stimulation. Ryanodine receptor 2 (RyR2) and (sarco)endoplasmic reticulum calcium ATPase 2a (SERCA2a) represent two critical targets of ß adrenoceptor (ßAR) signaling on the sarcoplasmic reticulum membrane for cardiac excitation and contraction coupling. Using novel biosensors, we show that cardiac ß1AR signals to both RyR2 and SERCA2a nanodomains in cardiomyocytes from mice, rats, and rabbits, whereas the ß2AR signaling is restricted from these nanodomains. Phosphodiesterase 4 (PDE4) and PDE3 control the baseline PKA activity and prevent ß2AR signaling from reaching the RyR2 and SERCA2a nanodomains. Moreover, blocking inhibitory G protein allows ß2AR signaling to the RyR2 but not the SERCA2a nanodomains. This study provides evidence for the differential roles of inhibitory G protein and PDEs in controlling the adrenergic subtype signaling at the RyR2 and SERCA2a nanodomains in cardiomyocytes. Video abstract.

GRK5 Controls SAP97-Dependent Cardiotoxic ß1 Adrenergic Receptor-CaMKII Signaling in Heart Failure.

RATIONALE: Cardiotoxic ß1 adrenergic receptor (ß1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of ß1AR and organizes a receptor signalosome. OBJECTIVE: We aim to elucidate the dynamics of ß1AR-SAP97 signalosome and its potential role in chronic cardiotoxic ß1AR-CaMKII signaling that contributes to development of heart failure. METHODS AND RESULTS: The integrity of cardiac ß1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine ß1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the ß1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of ß1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from ß1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of ß1AR-SAP97 complex and increases in CaMKII activity in hearts. CONCLUSIONS: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac ß1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Hyperinsulinemia promotes heterologous desensitization of ß2 adrenergic receptor in airway smooth muscle in obesity.

ß-Adrenergic receptor (ß-AR) agonists are the most common clinical bronchodilators for asthma. Obesity influences asthma severity and may impair response to ß-AR agonists. Previous studies show that in obese mice, hyperinsulinemia plays a crucial role in ß-AR desensitization in the heart. We therefore investigated whether insulin promotes ß-AR desensitization in airway smooth muscle (ASM) and compromises airway relaxation responsiveness to ß-AR agonists. We found that human ASM cells and mouse airway tissues exposed to insulin exhibit impaired ß2 AR-induced cAMP accumulation and airway relaxation. This impaired relaxation is associated with insulin-induced phosphorylation and expression of phosphodiesterase 4D (PDE4D) through transactivation of a G protein-coupled receptor kinase 2 (GRK2)-dependent ß2 AR-Gi -ERK1/2 cascade. Both acute and chronic pharmacological inhibition of PDE4 effectively reversed impaired ß2 AR-mediated ASM relaxation in an obesity mouse model induced by a high fat diet. Collectively, these findings reveal that cross talk between insulin and ß2 AR signaling promotes ASM ß2 AR desensitization in obesity through upregulation of PDE4D phosphorylation and expression. Our results identify a novel pathway of asthma pathogenesis in patients with obesity/metabolic syndrome, in which the GRK2-mediated signaling can be a potential therapeutic modality to prevent or treat ß2 AR desensitization in ASM. Moreover, PDE4 inhibitors may be used as efficacious therapeutic agents for asthma in obese and diabetic subjects.

Whole-Cell cAMP and PKA Activity are Epiphenomena, Nanodomain Signaling Matters.

Novel targeted fluorescent biosensors provide key insights into very local nanodomains of cAMP and PKA activity, and how they respond differently to ß-adrenergic activation in cardiac myocytes. This unique spatiotemporal detail in living cells is not available with biochemical measurements of total cellular cAMP and PKA, and provides unique physiological insights.

Phosphorylation of Cav1.2 on S1928 uncouples the L-type Ca2+ channel from the ß2 adrenergic receptor.

Agonist-triggered downregulation of ß-adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of ß2AR signaling highly specific for Cav1.2. We find that ß2-AR binding to Cav1.2 residues 1923-1942 is required for ß-adrenergic regulation of Cav1.2. Despite the prominence of PKA-mediated phosphorylation of Cav1.2 S1928 within the newly identified ß2AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the ß2AR from Cav1.2 upon ß-adrenergic stimulation rendering Cav1.2 refractory for several minutes from further ß-adrenergic stimulation. This effect is lost in S1928A knock-in mice. Although AMPARs are clustered at postsynaptic sites like Cav1.2, ß2AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the ß2AR from Cav1.2 is a uniquely specific desensitization mechanism of Cav1.2 regulation by highly localized ß2AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT-LTP) depends on Cav1.2 and its regulation by channel-associated ß2AR.

ß-adrenergic-mediated dynamic augmentation of sarcolemmal CaV 1.2 clustering and co-operativity in ventricular myocytes.

KEY POINTS: Prevailing dogma holds that activation of the ß-adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L-type CaV 1.2 channel activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood. CaV 1.2 channel clusters decorate T-tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by physical interactions between adjacent channel C-terminal tails. We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A-dependent augmentation of CaV 1.2 channel abundance along cardiomyocyte T-tubules, resulting in the appearance of channel 'super-clusters', and enhanced channel co-operativity that amplifies Ca2+ influx. On the basis of these data, we suggest a new model in which a sub-sarcolemmal pool of pre-synthesized CaV 1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation-contraction coupling. ABSTRACT: Voltage-dependent L-type CaV 1.2 channels play an indispensable role in cardiac excitation-contraction coupling. Activation of the ß-adrenergic receptor (ßAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced CaV 1.2 activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. CaV 1.2 channels exhibit a clustered distribution along the T-tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca2+ -dependent co-operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C-terminal tails. This amplifies Ca2+ influx and augments myocyte Ca2+ transient and contraction amplitudes. We investigated whether ßAR signalling could alter CaV 1.2 channel clustering to facilitate co-operative channel interactions and elevate Ca2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the ßAR agonist, isoproterenol (ISO), promotes enhanced CaV 1.2-CaV 1.2 physical interactions. Super-resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of CaV 1.2 'super-clusters' along the z-lines of ISO-stimulated cardiomyocytes. The mechanism that leads to super-cluster formation involves rapid, dynamic augmentation of sarcolemmal CaV 1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co-operative gating behaviour of CaV 1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of ßAR-mediated regulation of CaV 1.2 channels in the heart and support the novel concept that a pre-synthesized pool of sub-sarcolemmal CaV 1.2 channel-containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation-contraction coupling to meet metabolic and/or haemodynamic demands.

Inhibiting Insulin-Mediated ß2-Adrenergic Receptor Activation Prevents Diabetes-Associated Cardiac Dysfunction.

BACKGROUND: Type 2 diabetes mellitus (DM) and obesity independently increase the risk of heart failure by incompletely understood mechanisms. We propose that hyperinsulinemia might promote adverse consequences in the hearts of subjects with type-2 DM and obesity. METHODS: High-fat diet feeding was used to induce obesity and DM in wild-type mice or mice lacking ß2-adrenergic receptor (ß2AR) or ß-arrestin2. Wild-type mice fed with high-fat diet were treated with a ß-blocker carvedilol or a GRK2 (G-protein-coupled receptor kinase 2) inhibitor. We examined signaling and cardiac contractile function. RESULTS: High-fat diet feeding selectively increases the expression of phosphodiesterase 4D (PDE4D) in mouse hearts, in concert with reduced protein kinase A phosphorylation of phospholamban, which contributes to systolic and diastolic dysfunction. The expression of PDE4D is also elevated in human hearts with DM. The induction of PDE4D expression is mediated by an insulin receptor, insulin receptor substrate, and GRK2 and ß-arrestin2-dependent transactivation of a ß2AR-extracellular regulated protein kinase signaling cascade. Thus, pharmacological inhibition of ß2AR or GRK2, or genetic deletion of ß2AR or ß-arrestin2, all significantly attenuate insulin-induced phosphorylation of extracellular regulated protein kinase and PDE4D induction to prevent DM-related contractile dysfunction. CONCLUSIONS: These studies elucidate a novel mechanism by which hyperinsulinemia contributes to heart failure by increasing PDE4D expression and identify ß2AR or GRK2 as plausible therapeutic targets for preventing or treating heart failure in subjects with type 2 DM.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

RATIONALE: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. OBJECTIVE: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. METHODS AND RESULTS: We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is because of an enhanced ß2 adrenergic receptor signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of ß2 adrenergic receptor signal by preventing PKA signal access to the myofilaments and to restore contractile response to adrenergic stimulation. CONCLUSIONS: In hypertrophic rabbit myocytes, selectively enhanced ß2 adrenergic receptor signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine ß2 adrenergic receptor signaling and restore myocyte contractility in response to ß adrenergic stimulation.

High-fat diet induces protein kinase A and G-protein receptor kinase phosphorylation of ß2 -adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts.

KEY POINTS: Patients with diabetes show a blunted cardiac inotropic response to ß-adrenergic stimulation despite normal cardiac contractile reserve. Acute insulin stimulation impairs ß-adrenergically induced contractile function in isolated cardiomyocytes and Langendorff-perfused hearts. In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high-fat diet (HFD) feeding on the cardiac ß2 -adrenergic receptor signalling and the impacts on cardiac contractile function. We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to ß-adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in ß2 -adrenergic receptor phosphorylation at protein kinase A and G-protein receptor kinase sites in the myocardium. The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. ABSTRACT: Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high-fat diet (HFD) on the insulin-adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD-fed mice displayed a significant elevation of phosphorylation of the ß2 -adrenergic receptor (ß2 AR) at both the protein kinase A site serine 261/262 and the G-protein-coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD-fed mice also displayed a reduced contractile response to adrenergic stimulation when compared with those of control mice fed normal chow. Genetic deletion of the ß2 AR led to a normalized adrenergic response and preserved cardiac contractile reserve in HFD-fed mice. Together, these data indicate that HFD promotes phosphorylation of the ß2 AR, contributing to impairment of cardiac contractile reserve before cardiac structural and functional remodelling, suggesting that early intervention in the insulin-adrenergic signalling network might be effective in prevention of cardiac complications in diabetes.

Cross-Talk Between Insulin Signaling and G Protein-Coupled Receptors.

Diabetes is a major risk factor for the development of heart failure. One of the hallmarks of diabetes is insulin resistance associated with hyperinsulinemia. The literature shows that insulin and adrenergic signaling is intimately linked to each other; however, whether and how insulin may modulate cardiac adrenergic signaling and cardiac function remains unknown. Notably, recent studies have revealed that insulin receptor and ß2 adrenergic receptor (ß2AR) forms a membrane complex in animal hearts, bringing together the direct contact between 2 receptor signaling systems, and forming an integrated and dynamic network. Moreover, insulin can drive cardiac adrenergic desensitization via protein kinase A and G protein-receptor kinases phosphorylation of the ß2AR, which compromises adrenergic regulation of cardiac contractile function. In this review, we will explore the current state of knowledge linking insulin and G protein-coupled receptor signaling, especially ß-adrenergic receptor signaling in the heart, with emphasis on molecular insights regarding its role in diabetic cardiomyopathy.

Probing cellular protein complexes using single-molecule pull-down.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.

Genetic suppression of ß2-adrenergic receptors ameliorates tau pathology in a mouse model of tauopathies.

Accumulation of the microtubule-binding protein tau is a key event in several neurodegenerative disorders referred to as tauopathies, which include Alzheimer's disease, frontotemporal lobar degeneration, Pick's disease, progressive supranuclear palsy and corticobasal degeneration. Thus, understanding the molecular pathways leading to tau accumulation will have a major impact across multiple neurodegenerative disorders. To elucidate the pathways involved in tau pathology, we removed the gene encoding the beta-2 adrenergic receptors (ß2ARs) from a mouse model overexpressing mutant human tau. Notably, the number of ß2ARs is increased in brains of AD patients and epidemiological studies show that the use of beta-blockers decreases the incidence of AD. The mechanisms underlying these observations, however, are not clear. We show that the tau transgenic mice lacking the ß2AR gene had a reduced mortality rate compared with the parental tau transgenic mice. Removing the gene encoding the ß2ARs from the tau transgenic mice also significantly improved motor deficits. Neuropathologically, the improvement in lifespan and motor function was associated with a reduction in brain tau immunoreactivity and phosphorylation. Mechanistically, we provide compelling evidence that the ß2AR-mediated changes in tau were linked to a reduction in the activity of GSK3ß and CDK5, two of the major tau kinases. These studies provide a mechanistic link between ß2ARs and tau and suggest the molecular basis linking the use of beta-blockers to a reduced incidence of AD. Furthermore, these data suggest that a detailed pharmacological modulation of ß2ARs could be exploited to develop better therapeutic strategies for AD and other tauopathies.

A long lasting ß1 adrenergic receptor stimulation of cAMP/protein kinase A (PKA) signal in cardiac myocytes.

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 ß1 adrenergic receptor (ß1AR) 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 ß1AR-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. ß1AR 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 ß1AR 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.