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J Biol Chem. 2018 May 11;293(19):7466-7473. doi: 10.1074/jbc.RA118.001975. Epub 2018 Mar 9.

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

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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.
From the Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, Georgia 30912,


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.


BRET; G protein; G protein–coupled receptor (GPCR); NanoLuc; arrestin; biosensor; mini G protein; molecular pharmacology; protein complementation

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