Phosphodiesterase 4D is required for β
            <sub>2</sub>
            adrenoceptor subtype-specific signaling in cardiac myocytes

Xiang, Yang; Naro, Fabio; Zoudilova, Maria; Jin, S.-L. Catherine; Conti, Marco; Kobilka, Brian K.

doi:10.1073/pnas.0405263102

Cited by 116 publications

(142 citation statements)

References 33 publications

Supporting

Mentioning

131

Contrasting

Unclassified

Order By: Relevance

“…9) while having no effect on another G␣ s -coupled receptor (V 2 R). These findings better define the role of the PDE4 family, whose involvement in agonist-mediated ␤ 2 AR signaling had been described (42,43) and whose inhibitors are therapeutic targets for a variety of diseases (44). The existence of PDE4 and ␤ 2 AR in the same complex under basal conditions could explain these results (45,46).…”

Section: Discussionmentioning

confidence: 61%

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Stefan

Aquin²,

Berger³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

170

177

View full text Add to dashboard Cite

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-tobackground ratio to identify endogenously expressed G␣s proteincoupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive ␤-2 adrenergic-but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.G protein-coupled receptor ͉ complementation assays ͉ protein-protein interactions ͉ protein fragment G protein-coupled receptors (GPCRs) represent the largest family of cell-surface molecules involved in signal transmission. GPCRs play roles in a broad range of biological processes through regulating the majority of cell-to-cell and cell-toenvironment communication, and, consequently, their dysfunction manifests in numerous diseases (1, 2). The GPCR family has enormous pharmacological importance, as demonstrated by the fact that Ͼ30% of approved drugs elicit their therapeutic effect by selectively acting on known members of this family (3). The human genome harbors Ͼ800 putative GPCRs including a considerable number with unknown physiological function or ligands. GPCR cascades hence remain a major focus of molecular pharmacology (4, 5).Signal transduction by GPCRs is mediated by activation of protein kinases (4), among which the most intensively studied is the cAMP-dependent protein kinase A (PKA) (6). Various extracellular signals converge on the cAMP/PKA pathway through ligand binding to GPCRs. The adenylyl cyclase then converts ATP to the ubiquitous second messenger cAMP. Intrac...

show abstract

Section: Discussionmentioning

confidence: 61%

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Stefan

Aquin²,

Berger³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

170

177

View full text Add to dashboard Cite

show abstract

“…2+ levels and myocyte contraction rate after bAR stimulation It is well established that stimulation of b 1 AR signaling increases heart rate (Devic et al, 2001;Xiang et al, 2005) and that this effect is due in part to increased PKA-mediated phosphorylation and activation of Ca V 1.2 and RyR2 (Bers, 2008;Shan et al, 2010). The fact that PDE4B controls the PKA-mediated phosphorylation of LTCC and RyR2 prompted us to compare the intracellular Ca 2+ levels and the contraction rate of wild-type and PDE4BKO NCMs.…”

Section: Pde4b Ablation Increases Intracellular Camentioning

confidence: 99%

PDE4B mediates local feedback regulation of β1-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes

Mika

Richter

Westenbroek

et al. 2014

Journal of Cell Science

Self Cite

View full text Add to dashboard Cite

Multiple cAMP phosphodiesterase (PDE) isoforms play divergent roles in cardiac homeostasis but the molecular basis for their nonredundant function remains poorly understood. Here, we report a novel role for the PDE4B isoform in b-adrenergic (bAR) signaling in the heart. Genetic ablation of PDE4B disrupted bAR-induced cAMP transients, as measured by FRETsensors, at the sarcolemma but not in the bulk cytosol of cardiomyocytes. This effect was further restricted to a subsarcolemmal compartment because PDE4B regulates b 1 AR-, but not b 2 AR-or PGE2-induced responses. The spatially restricted function of PDE4B was confirmed by its selective effects on PKA-mediated phosphorylation patterns. PDE4B limited the PKA-mediated phosphorylation of key players in excitationcontraction coupling that reside in the sarcolemmal compartment, including L-type Ca 2+ channels and ryanodine receptors, but not phosphorylation of distal cytosolic proteins. b 1 AR-but not b 2 ARligation induced PKA-dependent activation of PDE4B and interruption of this negative feedback with PKA inhibitors increased sarcolemmal cAMP. Thus, PDE4B mediates a crucial PKAdependent feedback that controls b 1 AR-dependent cAMP signals in a restricted subsarcolemmal domain. Disruption of this feedback augments local cAMP/PKA signals, leading to an increased intracellular Ca 2+ level and contraction rate.

show abstract

“…2a, b). It was found before that pronounced β-adrenergic stimulation or moderate β-adrenergic stimulation combined with PDE inhibition causes excessive levels of cAMP in cardiac myocytes but fails to show equivalent increases in contraction responses (Xiang et al 2005;De Arcangelis et al 2008). This discrepancy can be explained by increased protein phosphatase (PP) activity in response to high levels of cAMP, which prevents a hyperphosphorylation of PKA target proteins and consequentially limits contraction responses (De Arcangelis et al 2008).…”

Section: Discussionmentioning

confidence: 99%

Influence of phosphodiesterases and cGMP on cAMP generation and on phosphorylation of phospholamban and troponin I by 5-HT4 receptor activation in porcine left atrium

Weninger

Maeyer

Lefebvre

2013

Naunyn-Schmiedeberg's Arch Pharmacol

View full text Add to dashboard Cite

Our objective was to investigate the role of phosphodiesterase (PDE)3 and PDE4 and cGMP in the control of cAMP metabolism and of phosphorylation of troponin I (TnI) and phospholamban (PLB) when 5-HT 4 receptors are activated in pig left atrium. Electrically paced porcine left atrial muscles, mounted in organ baths, received stimulators of particulate guanylyl cyclase (pGC) or soluble guanylyl cyclase (sGC) and/or specific PDE inhibitors followed by 5-HT or the 5-HT 4 receptor agonist prucalopride. Muscles were freezeclamped at different moments of exposure to measure phosphorylation of the cAMP/protein kinase A targets TnI and PLB by immunoblotting and cAMP levels by enzyme immunoassay. Corresponding with the functional results, 5-HT only transiently increased cAMP content, but caused a less quickly declining phosphorylation of PLB and did not significantly change TnI phosphorylation. Under combined PDE3 and PDE4 inhibition, the 5-HT-induced increase in cAMP levels and PLB phosphorylation was enhanced and sustained, and TnI phosphorylation was now also increased. Responses to prucalopride per se and the influence thereupon of PDE3 and PDE4 inhibition were similar except that responses were generally smaller. Stimulation of pGC together with PDE4 inhibition increased 5-HT-induced PLB phosphorylation compared to 5-HT alone, consistent with functional responses. sGC stimulation hastened the fade of inotropic responses to 5-HT, while cAMP levels were not altered. PDE3 and PDE4 control the cAMP response to 5-HT 4 receptor activation, causing a dampening of downstream signalling. Stimulation of pGC is able to enhance inotropic responses to 5-HT by increasing cAMP levels, while sGC stimulation decreases contraction to 5-HT cAMP independently.

show abstract

Phosphodiesterase 4D is required for β ₂ adrenoceptor subtype-specific signaling in cardiac myocytes

Cited by 116 publications

References 33 publications

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

PDE4B mediates local feedback regulation of β1-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes

Influence of phosphodiesterases and cGMP on cAMP generation and on phosphorylation of phospholamban and troponin I by 5-HT4 receptor activation in porcine left atrium

Contact Info

Product

Resources

About

Phosphodiesterase 4D is required for β 2 adrenoceptor subtype-specific signaling in cardiac myocytes

Cited by 116 publications

References 33 publications

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo

PDE4B mediates local feedback regulation of β1-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes

Influence of phosphodiesterases and cGMP on cAMP generation and on phosphorylation of phospholamban and troponin I by 5-HT4 receptor activation in porcine left atrium

Contact Info

Product

Resources

About

Phosphodiesterase 4D is required for β ₂ adrenoceptor subtype-specific signaling in cardiac myocytes