Persistent cAMP-Signals Triggered by Internalized G-Protein–Coupled Receptors

Calebiro, Davide; Nikolaev, Viacheslav O.; Gagliani, Maria Cristina; Filippis, Tiziana de; Dees, Christian; Tacchetti, Carlo; Persani, Luca; Lohse, Martin J.

doi:10.1371/journal.pbio.1000172

Cited by 519 publications

(556 citation statements)

References 91 publications

(117 reference statements)

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“…Next, we performed FRET recordings under selective stimulations of b 1 -and b 2 -AR in Epac1-PLN-expressing cardiomyocytes and compared local cAMP responses with those measured in the bulk cytosol by using previously established mice expressing the cytosolic Epac1-camps biosensor 19 . Interestingly, strong b 1 -AR-cAMP signals were present in both SERCA2a microdomain and bulk cytosol, while much smaller b 2 -ARcAMP responses were detectable only in the cytosol but not with Epac1-PLN sensor targeted to the SERCA2a microdomain ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Some other biosensors, such as the A-kinase activity reporter fused to PLN have recently been introduced into adult cardiomyocytes via adenoviral vectors to monitor local kinase activity 21 . Although we and others have successfully generated TG mice expressing some of the cytosolic FRET biosensors 19,[22][23][24] , it remained unclear whether targeted, microdomain-specific biosensors, which act in the functionally relevant subcellular locations, are also compatible with in vivo animal models. Here, we describe the generation and validation of the first TG mouse model, which expresses a targeted cAMP biosensor designed to dissect the cAMP signalling in the vicinity of an important calcium-handling protein SERCA2a.…”

Section: Discussionmentioning

confidence: 99%

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In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

et al. 2015

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3 0 ,5 0 -cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger that regulates physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors are developed and used in single cells, it is unclear whether such biosensors can be successfully applied in vivo, especially in the context of disease. Here, we describe a transgenic mouse model expressing a targeted cAMP sensor and analyse microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signalling in adult cardiomyocytes isolated from a healthy mouse heart and from an in vivo cardiac disease model. In particular, we uncover the existence of a phosphodiesterase-dependent receptor-microdomain communication, which is affected in hypertrophy, resulting in reduced b-adrenergic receptor-cAMP signalling to SERCA.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

et al. 2015

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“…Sustained cAMP and G S signaling by receptor internalization in early endosomes was originally reported in 2009 for two distinct GPCRs, the thyroid-stimulating hormone receptor (TSHR) (35) and PTHR (2), and recently expanded to the dopamine D1 receptor (D1R) (36). Endosomal G-protein signaling appears to be an alternative pathway not only for G S -but also for inhibitory G protein (Gi)-coupled receptors, as demonstrated for the sphingosine-1-phosphate receptor (37).…”

Section: Discussionmentioning

confidence: 96%

Noncanonical GPCR signaling arising from a PTH receptor–arrestin–Gβγ complex

Wehbi

Stevenson

Feinstein³

et al. 2013

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

159

107

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G protein-coupled receptors (GPCRs) participate in ubiquitous transmembrane signal transduction processes by activating heterotrimeric G proteins. In the current "canonical" model of GPCR signaling, arrestins terminate receptor signaling by impairing receptor-G-protein coupling and promoting receptor internalization. However, parathyroid hormone receptor type 1 (PTHR), an essential GPCR involved in bone and mineral metabolism, does not follow this conventional desensitization paradigm. β-Arrestins prolong G protein (G S )-mediated cAMP generation triggered by PTH, a process that correlates with the persistence of arrestin-PTHR complexes on endosomes and which is thought to be associated with prolonged physiological calcemic and phosphate responses. This presents an inescapable paradox for the current model of arrestin-mediated receptor-G-protein decoupling. Here we show that PTHR forms a ternary complex that includes arrestin and the Gβγ dimer in response to PTH stimulation, which in turn causes an accelerated rate of G S activation and increases the steady-state levels of activated G S , leading to prolonged generation of cAMP. This work provides the mechanistic basis for an alternative model of GPCR signaling in which arrestins contribute to sustaining the effect of an agonist hormone on the receptor.

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“…A rare example is a study by Calebiro et al (2009). They attempted to generate transgenic mice carrying a FRET biosensor for cAMP, Epac1-camp.…”

Section: Discussionmentioning

confidence: 99%

Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors

Kamioka

Sumiyama

Mizuno

et al. 2012

Cell Struct. Funct.

132

155

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ABSTRACT. Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

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Persistent cAMP-Signals Triggered by Internalized G-Protein–Coupled Receptors

Abstract: Real-time monitoring of G-protein-coupled receptor (GPCR) signaling in native cells suggests that the receptor for thyroid stimulating hormone remains active after internalization, challenging the current model for GPCR signaling.

Cited by 519 publications

References 91 publications

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

Noncanonical GPCR signaling arising from a PTH receptor–arrestin–Gβγ complex

Live Imaging of Protein Kinase Activities in Transgenic Mice Expressing FRET Biosensors

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