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Updated: Jun 23, 2025
09:12
G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay
Published on: September 10, 2016
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Conformation- and activation-based BRET sensors differentially report on GPCR-G protein coupling
Shane C Wright1,2,3, Charlotte Avet2, Supriya A Gaitonde1,2
1Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, QC H3T 1J4, Canada.
Science Signaling
|June 18, 2024
Summary
Different biosensor types reveal complexities in G protein-coupled receptor (GPCR) signaling. Experimental design, including G protein subunit choice and epitope tagging, critically impacts assessing GPCR-G protein coupling specificity.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- G protein-coupled receptors (GPCRs) mediate cellular responses through interactions with heterotrimeric G proteins (Gα, Gβ, Gγ).
- Bioluminescence resonance energy transfer (BRET) biosensors are valuable tools for studying GPCR functional selectivity and signaling.
- Existing BRET biosensors monitor either ligand-induced conformational changes or downstream effector recruitment.
Purpose of the Study:
- To compare conformation-based and activation-based BRET biosensors for assessing GPCR-Gα protein coupling.
- To investigate the influence of Gβγ subunits and epitope tags on GPCR coupling specificity.
Main Methods:
- Utilized BRET biosensors to monitor coupling of various class A and B GPCRs (5-HT2A, 5-HT7, GLP-1R, M3) to specific Gα proteins in cultured cells.
- Assessed the impact of different Gβγ subunit combinations and epitope tags on receptor-G protein interactions.
Main Results:
- Observed distinct signaling profiles between conformation- and activation-based BRET biosensors, affecting data interpretation.
- Demonstrated that Gβγ subunit identity differentially influences GPCR selectivity for Gα subtypes.
- Found that epitope tagging of receptors can alter stoichiometry, coupling selectivity, and produce artifactual results.
Conclusions:
- The choice of BRET biosensor type is critical for accurate assessment of GPCR-G protein coupling.
- Gβγ subunit composition plays a significant role in determining GPCR-Gα coupling specificity.
- Careful experimental design, including consideration of epitope tags, is essential to avoid artifacts when studying GPCR signaling.
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