NanoBRET Approaches to Study Ligand Binding to GPCRs and RTKs

• Published in: Trends in pharmacological sciences (Regular ed.) Vol. 39; no. 2; pp. 136 - 147
• Main Authors: Stoddart, Leigh A., Kilpatrick, Laura E., Hill, Stephen J.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.02.2018
• Subjects: Animals; bioluminescence; Bioluminescence Resonance Energy Transfer Techniques - methods; G-protein-coupled receptor; Humans; ligand binding; Ligands; NanoBRET; NanoLuc luciferase; Protein Binding; Receptor Protein-Tyrosine Kinases - chemistry; Receptor Protein-Tyrosine Kinases - metabolism; receptor tyrosine kinase; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - metabolism; Single Molecule Imaging - methods; NanoLuc luciferase; receptor tyrosine kinase; NanoBRET; bioluminescence; ligand binding; G-protein-coupled receptor
• ISSN: 0165-6147; 1873-3735
• DOI: 10.1016/j.tips.2017.10.006

Recent advances in the development of fluorescent ligands for G-protein-coupled receptors (GPCRs) and receptor tyrosine kinase receptors (RTKs) have facilitated the study of these receptors in living cells. A limitation of these ligands is potential uptake into cells and increased nonspecific binding. However, this can largely be overcome by using proximity approaches, such as bioluminescence resonance energy transfer (BRET), which localise the signal (within 10nm) to the specific receptor target. The recent engineering of NanoLuc has resulted in a luciferase variant that is smaller and significantly brighter (up to tenfold) than existing variants. Here, we review the use of BRET from N-terminal NanoLuc-tagged GPCRs or a RTK to a receptor-bound fluorescent ligand to provide quantitative pharmacology of ligand–receptor interactions in living cells in real time. Recent development of the luciferase variant NanoLuc and its use in conjunction with fluorescent ligand technologies has allowed the BRET-based quantification of ligand binding at a range of GPCRs. NanoBRET is a safer, more cost-effective alternative to radioligand binding. The inherent distance limits of BRET overcome issues of high levels of nonspecific binding often associated with radiolabelled, fluorescent, and lipophilic ligands. Traditional measures of ligand pharmacology, such as binding kinetics and affinity, can be measured in real time in live cells at a range of membrane-bound receptors using NanoBRET. The advantages of NanoBRET allow the phenomena of allosterism, probe dependence, and cooperativity to be studied in living cells. Quantitative pharmacology has been successfully determined at typically challenging receptors, such as fatty acid receptors and full-length vascular endothelial growth factor receptor 2 (VEGFR2).