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

Qingwen Wan, Najeah Okashah, Asuka Inoue, Rony Nehme, Byron Carpenter, Christopher G. Tate, Nevin A. Lambert

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 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 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 subtype–biased ligands.

Original languageEnglish (US)
Pages (from-to)7466-7473
Number of pages8
JournalJournal of Biological Chemistry
Volume293
Issue number19
DOIs
StatePublished - Jan 1 2018

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G-Protein-Coupled Receptors
GTP-Binding Proteins
Chemical activation
Bioluminescence
Assays
Energy Transfer
Luciferases
Energy transfer
Proteins
Endosomes
GTP Phosphohydrolases
Golgi Apparatus
Cell membranes
Luminescence
Organelles
Cytoplasm
Fusion reactions
Cells
Cell Membrane
Ligands

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Mini G protein probes for active G protein– coupled receptors (GPCRs) in live cells. / Wan, Qingwen; Okashah, Najeah; Inoue, Asuka; Nehme, Rony; Carpenter, Byron; Tate, Christopher G.; Lambert, Nevin A.

In: Journal of Biological Chemistry, Vol. 293, No. 19, 01.01.2018, p. 7466-7473.

Research output: Contribution to journalArticle

Wan, Qingwen ; Okashah, Najeah ; Inoue, Asuka ; Nehme, Rony ; Carpenter, Byron ; Tate, Christopher G. ; Lambert, Nevin A. / Mini G protein probes for active G protein– coupled receptors (GPCRs) in live cells. In: Journal of Biological Chemistry. 2018 ; Vol. 293, No. 19. pp. 7466-7473.
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