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      Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells

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          G protein–coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase–mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype–biased ligands.

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          Most cited references 26

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          A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.

          The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.
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            Seven-transmembrane receptors.

            Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors, are also the most common target of therapeutic drugs. Recent findings indicate that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions.
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              Is Open Access

              THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: G protein‐coupled receptors

              The Concise Guide to PHARMACOLOGY 2017/18 provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to an open access knowledgebase of drug targets and their ligands (, which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at G protein‐coupled receptors are one of the eight major pharmacological targets into which the Guide is divided, with the others being: ligand‐gated ion channels, voltage‐gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2017, and supersedes data presented in the 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

                Author and article information

                J Biol Chem
                J. Biol. Chem
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A. )
                11 May 2018
                9 March 2018
                9 March 2018
                : 293
                : 19
                : 7466-7473
                From the []Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, Georgia 30912,
                the [§ ]Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578 Japan, and
                the []MRC Laboratory of Molecular Biology, Cambridge CB20QH, United Kingdom
                Author notes
                [4 ] To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA 30912-2300. Tel.: 706-721-6336; Fax: 706-721-2345; E-mail: nelambert@ .

                Supported by Japan Science and Technology Agency (JST) Grant JPMJPR1331 and Japan Agency for Medical Research and Development (AMED) Grant JP17gm5910013.


                Present address: Creoptix AG, Einsiedlerstrasse 34, CH-8820 Wädenswil, Switzerland.


                Present address: Warwick Integrative Synthetic Biology Centre, University of Warwick, Coventry CV4 7AL, United Kingdom.

                Edited by Henrik G. Dohlman

                © 2018 Wan et al.

                Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version free via Creative Commons CC-BY license.

                Funded by: HHS | National Institutes of Health (NIH) , open-funder-registry 10.13039/100000002;
                Award ID: GM078319
                Award ID: GM109879
                Funded by: RCUK | Medical Research Council (MRC) , open-funder-registry 10.13039/501100000265;
                Award ID: MC_U105197215
                Funded by: EC | FP7 | FP7 Ideas: European Research Council (FP7 Ideas) , open-funder-registry 10.13039/100011199;
                Award ID: EMPSI 339995
                Funded by: MEXT | Japan Science and Technology Agency (JST) , open-funder-registry 10.13039/501100002241;
                Award ID: JPMJPR1331
                Funded by: Japan Agency for Medical Research and Development (AMED) , open-funder-registry 10.13039/100009619;
                Award ID: JP17gm5910013
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