G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

Summary

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, Fӧrster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

Abstract

Fӧrster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between Gα subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the β₂-adrenergic receptor and the Gαs C-terminus peptide upon treatment with 100 µM isoproterenol hydrochloride as previously characterized¹. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a Gα C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Introduction

G-protein-coupled receptors (GPCRs) are seven-transmembrane receptors. The human genome alone contains approximately 800 genes coding for GPCRs, which are activated by a variety of ligands including light, odorants, hormones, peptides, drugs and other small molecules. Nearly 30% of all pharmaceuticals currently on the market target GPCRs because they play a large role in many disease states². Despite decades of extensive work done on this receptor family, there remain significant outstanding questions in the field, particularly with regards to the molecular mechanisms that drive GPCR-effector interactions. To date, only one high-resolution crystal structure....

Protocol

1. DNA Preparation

1. Design sensor constructs using a modular cloning scheme. Please reference the β₂-AR sensor design detailed previously¹.
2. Prepare DNA according to commercial miniprep kit protocol and elute in 2 mM Tris-HCl solution, pH 8, at concentration ≥ 750 ng/µl, A₂₆₀/A₂₈₀ of 1.7 - 1.9, A₂₆₀/A₂₃₀ of 2.0 - 2.29.

2. Cell Culture Preparation

1. Culture HEK-293T-Flp-n cells in DMEM containing 4.5 g/L D-glucose, supplemented with 10% FBS (heat inactivated) (v/v), 1% L-glutamine supplement, 20 mM HEPES, pH 7.5 at 37 °C in humidified atmos....

Results

A generalized schematic of the experiment set up and execution is detailed in Figure 3.

In order to detect a FRET change in the narrow dynamic range of the sensor, it is critical to adhere to the nuances of the system be adhered to. Cell quality is imperative to protein expression as well as consistency in sampling. Figure 1 features images of cultured cells growing in a consistent monolayer (10X) that is optimal for six-well plating and transfection F.......

Discussion

The tight dynamic range of FRET measurements in this system reinforces the necessity of sensitive quality control in every step of this protocol. The most important steps to ensure a successful FRET experiment are 1) cell culturing, 2) transfection 3) protein expression and 4) timely, precise coordination during the assay execution.

Cell health and maintenance/plating quality can have a significant impact on the signal-to-noise of the experimental system and poor cell health can make it imposs.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
B2-AR-10 nm-Gas peptide sensor	Addgene	47438	https://www.addgene.org/Sivaraj_Sivaramakrishnan/
GeneJET Plasmid Miniprep Kit	Fermentas/Fisher Sci	FERK0503	Elute in 2 mM Tris elution buffer
HEK-293T-Flp-n cells	Life Technologies	R78007
Trypsin (0.25%)	Life Technologies	25200056
DMEM- high glucose	Life Technologies	11960-044	Warm in  37 °C water bath before use
FBS, certified, Heat inactivated, US origin	Life Technologies	10082147
Glutamax I 100x	Life Technologies	35050061
HEPES	Corning	MT25060CL
Opti-MEM	Life Technologies	31985-070	Reduced serum media; Bring to RT before use
XtremeGene HP transfection reagenet	Roche	6366236001	Highly recommended for its consistency. Bring to RT before use
FluoroMax 4	Horiba		Use with FluorEssence V3.8 software
3-mm path length quartz cuvette	Starna	NC9729944(16.45F-Q-3/z8.5)	May require cuvette holder/adaptor for use in Fluorometer, available from Starna
Sc100-S3 Heated Circulating water bath pump	Fisher Scientific	13-874-826	Warm to 37 °C before use
Thermomixer Heat Block	Eppendorf	22670000	Warm to 37 °C before use
Ultrapure DNA/RNAse free water	Life Technologies	10977015	Use at RT
D(+)-glucose, anhydrous	Sigma	G5767
aprotinin from bovine lung	Sigma	A1153
leupeptin hemisulfate	EMD	10-897
L-ascorbic acid, reagent grade	Sigma	A0278
(-)-isoproterenol (+)-bitartrate	Sigma	I2760	Use fresh aliquot each experiment