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Mini G protein probes for active G protein– coupled receptors (GPCRs) in live cells
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Wan, Qingwen, Okashah, Najeah, Inoue, Asuka, Nehmé, Rony, Carpenter, Byron, Tate, Christopher G. and Lambert, Nevin A. (2018) Mini G protein probes for active G protein– coupled receptors (GPCRs) in live cells. Journal of Biological Chemistry, 293 . pp. 7466-7473. doi:10.1074/jbc.RA118.001975
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WRAP-Mini-G-protein-coupled-receptors-Carpenter-2018.pdf - Publisher's Proof Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (1512Kb) | Preview |
Official URL: http://dx.doi.org/10.1074/jbc.RA118.001975
Abstract
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 G subunits that were developed for struc- tural 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 organ- elles. 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 G 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 sub- type–biased ligands.
| Item Type: | Journal Article | ||||||||||||||||||||||||
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| Subjects: | Q Science > QD Chemistry Q Science > QH Natural history > QH301 Biology |
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| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||||||||||||||||||||
| Library of Congress Subject Headings (LCSH): | G proteins, Ligand binding (Biochemistry) | ||||||||||||||||||||||||
| Journal or Publication Title: | Journal of Biological Chemistry | ||||||||||||||||||||||||
| Publisher: | American Society for Biochemistry and Molecular Biology | ||||||||||||||||||||||||
| ISSN: | 1083-351X | ||||||||||||||||||||||||
| Official Date: | 11 May 2018 | ||||||||||||||||||||||||
| Dates: |
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| Volume: | 293 | ||||||||||||||||||||||||
| Page Range: | pp. 7466-7473 | ||||||||||||||||||||||||
| DOI: | 10.1074/jbc.RA118.001975 | ||||||||||||||||||||||||
| Status: | Peer Reviewed | ||||||||||||||||||||||||
| Publication Status: | Published | ||||||||||||||||||||||||
| Access rights to Published version: | Open Access | ||||||||||||||||||||||||
| Copyright Holders: | American Society for Biochemistry and Molecular Biology | ||||||||||||||||||||||||
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