Time-resolved fluorescence ligand binding for G protein-coupled receptors

Area of Science:

• Biochemistry
• Pharmacology
• Molecular Biology

Background:

• Traditional radioligand-binding assays for G protein-coupled receptors (GPCRs) offer quantitative data but lack temporal/spatial resolution and pose safety risks.
• There is a need for alternative methods with improved resolution and safety profiles for characterizing GPCR-ligand interactions.

Purpose of the Study:

• To present a generalizable protocol for studying GPCRs and their ligands using time-resolved fluorescence resonance energy transfer (TR-FRET).
• To detail a method adaptable for various receptors and ligands, exemplified by the parathyroid hormone receptor.

Main Methods:

• Utilized terbium (Tb(+))-labeled fluorescent ligands and TR-FRET for high temporal and spatial resolution.
• Developed three protocol variations: ligand separation, SNAP-tag labeled receptors for high-throughput screening, and antibody-based detection with engineered epitope.

Main Results:

• Achieved high signal-to-background ratios across all presented protocol options.
• Demonstrated the ability to characterize ligands and receptors in living cells and cell membranes using plate-reader measurements.

Conclusions:

• The TR-FRET approach provides a safer, high-resolution alternative to radioligand assays for GPCR characterization.
• The presented protocols are versatile and can be readily adapted for diverse receptor-ligand systems in various biological contexts.