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

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          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.


          G-protein–coupled receptors (GPCRs) are generally thought to signal to second messengers like cyclic AMP (cAMP) from the cell surface and to become internalized upon repeated or prolonged stimulation. Once internalized, they are supposed to stop signaling to second messengers but may trigger nonclassical signals such as mitogen-activated protein kinase (MAPK) activation. Here, we show that a GPCR continues to stimulate cAMP production in a sustained manner after internalization. We generated transgenic mice with ubiquitous expression of a fluorescent sensor for cAMP and studied cAMP responses to thyroid-stimulating hormone (TSH) in native, 3-D thyroid follicles isolated from these mice. TSH stimulation caused internalization of the TSH receptors into a pre-Golgi compartment in close association with G-protein α s-subunits and adenylyl cyclase III. Receptors internalized together with TSH and produced downstream cellular responses that were distinct from those triggered by cell surface receptors. These data suggest that classical paradigms of GPCR signaling may need revision, as they indicate that cAMP signaling by GPCRs may occur both at the cell surface and from intracellular sites, but with different consequences for the cell.

          Author Summary

          Cells respond to many environmental cues through the activity of cell surface receptor proteins, which sense these cues and convey that information to signaling molecules inside the cell. G-protein–coupled receptors (GPCRs) form the largest eukaryotic family of plasma membrane receptors. They convert the information provided by extracellular stimuli into intracellular second messengers, like cyclic AMP (cAMP). After prolonged stimulation, they are internalized inside cells, an event that to date has been thought to terminate the production of second messengers. Though many of the key steps of GPCR signaling are known in detail, precisely how signaling and termination actually occur in time and space (i.e., in subcellular compartments or microdomains) is still largely unexplored. To observe GPCR signaling in living cells, we generated mice expressing a fluorescent sensor that allows monitoring the intracellular levels of cAMP with a microscope. We utilized this system to study, directly in native thyroid follicles, the signal sent by the receptor for thyroid-stimulating hormone (TSH). Our findings indicate that TSH receptors are internalized rapidly after activation but continue to stimulate cAMP production inside cells and that this sustained, cAMP production is apparently required for localized activation of downstream components. These data challenge the current model of the GPCR-cAMP pathway by suggesting the existence of previously unrecognized intracellular site(s) for cAMP generation and of differential signaling outcomes as a result of intracellular GPCR signaling. Such intracellular site(s) may provide specialized signaling platforms, thus contributing to the spatiotemporal regulation of cAMP production and to signaling specificity within the GPCR family.

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          Most cited references 80

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          AKAP signalling complexes: focal points in space and time.

          Multiprotein signalling networks create focal points of enzyme activity that disseminate the intracellular action of many hormones and neurotransmitters. Accordingly, the spatio-temporal activation of protein kinases and phosphatases is an important factor in controlling where and when phosphorylation events occur. Anchoring proteins provide a molecular framework that orients these enzymes towards selected substrates. A-kinase anchoring proteins (AKAPs) are signal-organizing molecules that compartmentalize various enzymes that are regulated by second messengers.
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            Novel single chain cAMP sensors for receptor-induced signal propagation.

            cAMP is a universal second messenger of many G-protein-coupled receptors and regulates a wide variety of cellular events. cAMP exerts its effects via cAMP-dependent protein kinase (PKA), cAMP-gated ion channels, and two isoforms of exchange protein directly activated by cAMP (Epac). Here we report the development of novel fluorescent indicators for cAMP based on the cAMP-binding domains of Epac and PKA. Fluorescence resonance energy transfer between variants of green fluorescent protein (enhanced cyan fluorescent protein and enhanced yellow fluorescent protein) fused directly to the cAMP-binding domains was used to analyze spatial and temporal aspects of cAMP-signaling in different cells. In contrast to previously developed PKA-based indicators, these probes are comprised of only a single binding site lacking cooperativity, catalytic properties, and interactions with other proteins and thereby allow us to easily image free intracellular cAMP and rapid signaling events. Rapid beta-adrenergic receptor-induced cAMP signals were observed to travel with high speed ( approximately 40 microm/s) throughout the entire cell body of hippocampal neurons and peritoneal macrophages. The developed indicators could be ubiquitously applied to studying cAMP, its physiological role and spatio-temporal regulation.
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              Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes.

              The second messenger cyclic adenosine monophosphate (cAMP) is the most important modulator of sympathetic control over cardiac contractility. In cardiac myocytes and many other cell types, however, cAMP transduces the signal generated upon stimulation of various receptors and activates different cellular functions, raising the issue of how specificity can be achieved. In the general field of signal transduction, the view is emerging that specificity is guaranteed by tight localization of signaling events. Here, we show that in neonatal rat cardiac myocytes, beta-adrenergic stimulation generates multiple microdomains with increased concentration of cAMP in correspondence with the region of the transverse tubule/junctional sarcoplasmic reticulum membrane. The restricted pools of cAMP show a range of action as small as approximately 1 micrometer, and free diffusion of the second messenger is limited by the activity of phosphodiesterases. Furthermore, we demonstrate that such gradients of cAMP specifically activate a subset of protein kinase A molecules anchored in proximity to the T tubule.

                Author and article information

                Role: Academic Editor
                PLoS Biol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                August 2009
                August 2009
                18 August 2009
                : 7
                : 8
                [1 ]Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany
                [2 ]Rudolf Virchow Center, DFG-Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany
                [3 ]Dipartimento di Scienze Mediche, Università degli Studi di Milano, Milan, Italy
                [4 ]Laboratory of Experimental Endocrinology, Fondazione IRCSS Istituto Auxologico Italiano, Cusano Milanino, Italy
                [5 ]Department of Experimental Medicine, University of Genoa, Genoa, Italy
                University of California San Francisco, United States of America
                Author notes

                The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: DC. Analyzed the data: DC CT LP. Contributed reagents/materials/analysis tools: VON LP. Wrote the paper: DC MJL. Isolated thyroid follicles; performed FRET, TIRF, immunofluorescence and cell fractionation experiments; generated the mathematical model: DC. Generated and analyzed the CAG-Epac1-camps mice; performed the FRET experiments on MEFs, cortical neurons, macrophages, and cardiac myocytes; performed the Western blot analysis of VASP phosphorylation: VON. Performed the electron microscopy experiments: MCG. Performed cell fractionation and Western blot experiments: TdF. Produced the fluorescently labeled TSH: CD. Supervised the electron microscopy experiments: CT. Supervised the project: MJL.

                Calebiro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 25
                Research Article
                Cell Biology/Cell Signaling
                Diabetes and Endocrinology/Thyroid

                Life sciences


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