Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• G protein-coupled receptors (GPCRs) are crucial cell surface receptors involved in numerous physiological processes.
• Measuring endogenous GPCR activity in primary cells presents significant experimental challenges.
• Existing methods may lack the versatility to detect all major G protein signaling pathways.

Purpose of the Study:

• To present a detailed protocol for utilizing ONE vector G protein Optical (ONE-GO) biosensors.
• To enable the measurement of endogenous GPCR activity across all four major G protein families (Gs, Gi/o, Gq/11, G12/13).
• To provide a versatile tool for studying GPCR signaling in primary cells.

Main Methods:

• Lentiviral transduction for expressing ONE-GO biosensors in primary cells.
• Bioluminescence Resonance Energy Transfer (BRET) assays for quantifying GPCR activity.
• Analysis of BRET signals to report on endogenous G protein activation.

Main Results:

• Successful expression of ONE-GO biosensors in primary cells.
• Quantification of endogenous GPCR activity through BRET measurements.
• Demonstration of the biosensors' ability to detect signaling through all four G protein families.

Conclusions:

• ONE-GO biosensors offer a versatile and effective method for studying endogenous GPCR activity in primary cells.
• This protocol facilitates high-throughput screening and detailed mechanistic studies of GPCR signaling.
• The approach enhances the understanding of GPCR function in physiological and pathological contexts.