Mini G protein probes for active G protein–coupled receptors (GPCRs) in live cells
| Autor: | Qingwen Wan, Christopher G. Tate, Rony Nehmé, Najeah Okashah, Byron Carpenter, Asuka Inoue, Nevin A. Lambert |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
G protein Endosome biosensor Biochemistry Receptors G-Protein-Coupled QH301 03 medical and health sciences 0302 clinical medicine GTP-Binding Proteins Arrestin Humans Editors' Picks QD Luciferase Luciferases Receptor Molecular Biology G protein-coupled receptor Binding Sites Microscopy Confocal NanoLuc arrestin Chemistry molecular pharmacology G protein–coupled receptor (GPCR) Cell Biology Fusion protein mini G protein Cell Compartmentation Cell biology HEK293 Cells 030104 developmental biology Energy Transfer Cytoplasm Molecular Probes Mutation BRET 030217 neurology & neurosurgery Protein Binding protein complementation |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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