Quantitative assessment of constitutive G protein-coupled receptor activity with BRET-based G protein biosensors

Abstract

Heterotrimeric G proteins constitute the primary transducers of G protein-coupled receptor (GPCR) signaling. Besides mediating ligand-induced GPCR activation, G proteins transduce basal levels of activity in various physiological and pathophysiological settings evoked by constitutively active, native GPCRs or disease-related receptor mutants. Several generations of optical biosensors were developed and optimized to monitor GPCR ligand-induced G protein activation, however, quantitative approaches to detect constitutively active GPCRs are not available. Here, we designed and validated a set of eight bioluminescence-resonance-energy-transfer (BRET)-based G protein sensors, covering all four major families of G proteins, and established a protocol to identify constitutive GPCR/G protein signaling in living cells. These sensors rely on the encoding of all three G protein subunits on a single plasmid, enabling their cellular expression at desired relative levels and resulting in reduced signal variability in mammalian cells. Based on this sensor platform, we further present here an experimental protocol to quantify constitutive signaling of native and mutated GPCRs through these heterotrimeric transducers. This approach will aid in the characterization of constitutively active GPCRs and the exploration of their role in health and disease.

A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3 Receptor

“…The G i2 BRET sensor was generated as previously described. 37 For the BRET measurements, HEK293A cells were transiently transfected in a suspension with wild-type H 3 R and G i2 BRET sensor (1:1 plasmid ratio) using Lipofectamine 2000 (Thermo Fisher Scientific, Nidderau, Germany) (2 μL transfection reagent/μg total plasmid) and seeded onto poly-D-lysine-precoated, white 96-well plates (Greiner Bio-One, Frickenhausen, Germany) (30,000 cells/well). 48 h after transfection, cells were washed with HBSS (Gibco/Life Technologies, Carlsbad, USA) and incubated with a 1/1000 stock solution of furimazine (Promega, Mannheim, Germany).…”

“…Therefore, we employed a recently developed BRET-based G i2 biosensor detecting G protein activation as a decrease in BRET between NLuc-tagged G αi2 and cpVenus-tagged G γ2 . 37 We first determined the potency of the selective H 3 R agonist imetit using the G i2 biosensor and HEK293A cells overexpressing the wild-type H 3 R (Figure S3, SI). 37 As expected, 12 acted as a neutral antagonist (Figure 3A) revealing a pK b value of 9.04 ± 0.03 in competition experiments with the selective H 3 R agonist imetit (Figure 3B).…”

“…37 We first determined the potency of the selective H 3 R agonist imetit using the G i2 biosensor and HEK293A cells overexpressing the wild-type H 3 R (Figure S3, SI). 37 As expected, 12 acted as a neutral antagonist (Figure 3A) revealing a pK b value of 9.04 ± 0.03 in competition experiments with the selective H 3 R agonist imetit (Figure 3B). However, before using 12 as a molecular tool to characterize other H 3 R ligands, the kinetic behavior of the fluorescent ligand should be determined first.…”

Identification of TSPAN4 as Novel Histamine H4 Receptor Interactor

“…HA-H 4 R in pcDEF3 was previously described [39]. Gα i2 protein biosensor plasmid was kindly provided by Dr. Schihada (Karolinska Institutet, Department of Physiology and Pharmacology, Stockholm, Sweden) [40]. All constructs were verified by DNA sequencing.…”

Functional modulation of PTH1R activation and signalling by RAMP2

“…To test this hypothesis, we used a suite of BRET-and FRET-based biosensors to quantify the effect of PTH1R activation on different signalling pathways, using both PTH and PTHrP. We first compared the ability of PTH1R to activate different G proteins, using BRET-based biosensors 38 , which respond to PTH1R activation with a decrease in BRET between their Gγ subunit labelled with the bioluminescent donor NanoLuc and their Gα subunits tagged with the acceptor cpVenus (Fig. 5).…”