Conformation- and Activation-Based BRET Sensors Differentially Report on GPCR-G Protein Coupling
Differential Study of GPCR-G Protein Coupling Biosensors
Background Introduction
G protein-coupled receptors (GPCRs) are important molecules for transmembrane signal transduction, capable of selectively binding to heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits and regulating various intracellular signaling processes. Studying the functional selectivity of GPCRs is crucial for understanding their biological functions and developing new drugs. However, the selective coupling between GPCRs and G proteins is not always straightforward. Different ligands can bias the receptor towards different Gα protein family subtypes. To study this complexity, bioluminescence resonance energy transfer (BRET) technology has been widely used to develop various biosensors for monitoring GPCR-G protein interactions. Nevertheless, different types of BRET biosensors may report coupling events differently.
Source of Study
This study, conducted by Shane C. Wright and colleagues, was published on June 18, 2024, in Science Signaling. The authors hail from several research institutions, including Université de Montréal, Karolinska Institutet, and the University of the Basque Country. The research team explored the differences in reporting GPCR-G protein coupling using BRET biosensors based on conformational changes and activation states.
Research Process
1. Research Methods and Subjects
The study primarily compared two types of BRET biosensors: Gαβγ sensors based on conformational changes and GEMTA sensors based on activation states. The research subjects included various GPCRs, such as 5-HT2A and 5-HT7 receptors, GLP-1 receptor (GLP-1R), and the M3 muscarinic receptor. These receptors were expressed in cultured cells, and their coupling with specific Gα proteins was monitored using different BRET sensors.
2. Experimental Design
- Gαβγ Sensor: By co-expressing a Renilla luciferase (Rluc)-tagged Gα subunit, an untagged Gβ subunit, and a GFP-tagged Gγ subunit, the conformational changes of the G protein trimer were monitored. Upon agonist addition, GPCR activation caused Gγ and Gβ subunits to dissociate from Gα-Rluc, leading to a decrease in BRET signal.
- GEMTA Sensor: Using engineered G protein effector proteins (like Rap1GAP1a and P63RhoGEF) combined with Rluc and membrane-anchored RGFP, the activation state of the Gα subunit post-receptor activation was monitored, manifesting as an increase in BRET signal.
3. Data Analysis
By comparing the responses of different BRET sensors in detecting GPCR-G protein coupling, the study evaluated the impact of sensor selection, receptor construction, and Gβγ subunit selection on experimental outcomes.