Development of a HiBiT Peptide-Based NanoBRET Ligand Binding Assay for Galanin Receptor 1 in Live Cells
This study investigates the development of a HiBiT peptide-based NanoBRET® assay to monitor ligand interactions with galanin receptor 1 (GALR1) in living cells. Galanin is a neuroendocrine peptide regulating physiological processes such as feeding, mood, anxiety, and pain modulation through its receptors GALR1, GALR2, and GALR3.
Traditional radioligand assays have limitations including radioactivity handling, poor kinetic resolution, and incompatibility with high-throughput screening. To address these issues, fluorescent peptide tracers derived from galanin peptides were developed, enabling real-time measurement of ligand binding kinetics in a physiologically relevant environment.
Results demonstrated strong correlation between NanoBRET® binding affinities and traditional radioligand binding assays. Additionally, real-time kinetic analyses effectively captured ligand interactions correlating with receptor internalization and β-arrestin recruitment activities.
This highlights the NanoBRET® assay as a powerful, physiologically relevant tool for live-cell assessment of GPCR-ligand interactions, offering improved resolution over traditional methods.
Keywords: NanoBRET, HiBiT peptide, galanin receptor, ligand-binding assay, GPCR, live cell imaging, BODIPY fluorophore, β-arrestin, receptor internalization, receptor kinetics, cell-based assay, bioluminescence imaging.
