A genetically encoded, fluorescent indicator for cyclic AMP in living cells

Zaccolo, Manuela; Giorgi, Francesca De; Cho, C Y; Feng, Lu; Knapp, Thomas E.; Negulescu, Paul A.; Taylor, Susan S.; Tsien, Roger Y.; Pozzan, Tullio

doi:10.1038/71345

Cited by 450 publications

(291 citation statements)

References 13 publications

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“…Immediately after isoproterenol treatment we observed the dissociation of the PKA reporter (t 1/2 ϭ 30.47 Ϯ 3.03 seconds). A similar pattern and timeframe of PKA activation has been observed with fluorescence resonance energy transfer-based PKA activity reporters in different cell types (10,11). Pretreatment of U2OS cells with the nonselective ␤-adrenergic antagonist alprenolol and with the selective ␤-2 adrenergic antagonist ICI118,551 prevented isoproterenol-mediated PKA activation to the same extent (Fig.…”

Section: Use Of the Pka Sensor To Monitor Protein Complex Dynamics Insupporting

confidence: 48%

“…Several cell-based assays have been developed to detect specific activation of PKA, including fluorescence and bioluminescent resonance energy transfer assays for detecting catalytic activity (10) or cAMP-induced PKA subunit dissociation (11)(12)(13). These methods have been invaluable to the study of protein complex dynamics and particularly to the integration of compartmentalized GPCR signaling pathways (14)(15)(16).…”

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confidence: 99%

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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.

174

179

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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...

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Section: Use Of the Pka Sensor To Monitor Protein Complex Dynamics Insupporting

confidence: 48%

mentioning

confidence: 99%

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.

174

179

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“…However, use of these probes in vivo is very limited because of the difficulty in their delivery into the cells. The development of a pair of GFP mutants that served as donor and acceptor overcame this problem and generated in vivo sensors for calcium (27), cAMP (28), and cGMP (29) or for monitoring the activities of protein kinase A (30), Rac (31), and Ras (32). After these successes, the development of Picchu showed that GFP-based FRET technology can be applied to the detection of the conformational change induced by tyrosine phosphorylation.…”

Section: Monitoring In Vivo Of Crkii Phosphorylation By Picchu-we Nexmentioning

confidence: 99%

A Pair of Fluorescent Resonance Energy Transfer-based Probes for Tyrosine Phosphorylation of the CrkII Adaptor Protein in Vivo

Kurokawa¹,

Mochizuki²,

Ohba³

et al. 2001

Journal of Biological Chemistry

144

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“…Further work is necessary to investigate if this "complementation-induced" BRET system can be useful for engineering intramolecular BRET biosensors, ( i.e. proteins carrying BRET labels tethered at distal ends of the molecule, so that the binding of signaling intermediates, like Ca 2+ or cAMP, can induce conformational changes that are reported as changes of RET efficiency [41][42][43][44][45]). One clear application, however, is the utilization of this Renilla system for engineering single-chain optical sensors of proteolysis [46].…”

Section: Obviously More Detailed Investigations Are Necessary To Vermentioning

confidence: 99%

Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells

Molinari

Casella

Costa

2007

Biochemical Journal

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Green bioluminescence in Renilla species is generated by a ∼100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein–protein interactions. To address the question, we compared and analysed in mammalian cells the bioluminescence of Rluc and RGFP co-expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled coils. Here, we show that: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. Here, we propose a new functional complementation assay based on the detection of the high-efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance, we implemented the assay to measure the interaction between GPCRs (G-protein-coupled receptors) and β-arrestins. We show that complementation-induced BRET allows detection of the GPCR–β-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.

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A genetically encoded, fluorescent indicator for cyclic AMP in living cells

Cited by 450 publications

References 13 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

A Pair of Fluorescent Resonance Energy Transfer-based Probes for Tyrosine Phosphorylation of the CrkII Adaptor Protein in Vivo

Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells

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