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

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.

Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

“…To increase Gs coupling, membrane of cells expressing Cys‐CRF1R mutants were treated with recombinant mini‐Gs; a truncated variant of Gαs . It has been shown that GPCRs bound to mini‐Gs proteins adopt similar active‐state conformations as those of Gs‐protein bound receptors . Indeed, yields of crosslinking on ClAc‐ligand/Cys‐CRF1R pairs increased by increasing the concentration of mini‐Gs in the probes (Figure A).…”

Differences in signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide

“…At the end of the incubation period, cells were lysed, and cAMP determined by HTRF (cAMP Dynamic 2 kit, Cisbio) using a Spectramax i3x plate reader (Molecular Devices). The plasmids for mini-G s , -G i and -G q , each tagged at the N-terminus with the LgBiT tag (Wan et al, 2018), were a kind gift from Prof Nevin Lambert, Medical College of Georgia.…”

Making useful gadgets with miniaturized G proteins

“…Using this strategy, the study beautifully documents detection of GPCR activation at various subcellular locations including plasma membrane, the Golgi apparatus, and endosomes (8). More quantitative and kinetic information on GPCR activation can be obtained when mGs and GPCRs are tagged with BRET pairs, yielding robust and reproducible ratiometric measurements in multiwell assay format on plate readers (8). What if you do not have access to sophisticated instruments or feel uncomfortable doing live-cell microscopy or BRET measurements?…”

“…In this issue of JBC, Nevin Lambert's group describes the development of a new arsenal of tools and assays to monitor GPCR activation directly and robustly using standard equipment available in most laboratories (8). The new technology is based on repurposing the so-called "mini G proteins" (mGs) originally developed as structural biology tools by Carpenter, Tate, and colleagues (9, 10), who co-authored this paper.…”

“…1). In their report, Wan et al (8) show that mGs fused with reporter probes can be used to quantify GPCR activation in multiple cell-based assay formats (see below). As long as the GPCR is active, the mG remains bound to it, thereby reporting the activation.…”