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 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.
Highlights
G protein– coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function
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 proteins were originally engineered for high-level expression in Escherichia coli, high stability in vitro, and effective coupling to GPCRs
Summary
Mini G proteins were originally engineered for high-level expression in Escherichia coli, high stability in vitro, and effective coupling to GPCRs. Stimulation of 2-adrenergic receptors fused to the fluorescent protein cerulean (2AR– cerulean) with a saturating concentration of the agonist isoproterenol resulted in rapid translocation of NES–venus–mGs from the cytosol to the plasma membrane (Fig. 1, B and C). To estimate the stoichiometry of receptor–mG complexes at steady state, we used standardized confocal imaging conditions to measure cerulean and venus fluorescence at the plasma membrane of cells expressing cerulean-2AR and venus–mGs in the presence of 10 M isoproterenol. We calibrated these intensity measurements with a standard protein consisting of an extracellular cerulean, a transmembrane domain, and an intracellular venus (C-TM-V). There was detectable accumulation of mG proteins at the plasma membrane prior to stimulation (Fig. 1D), suggesting that mG proteins could bind to ligand-free GPCRs (see below)
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