The NanoBiT-Based Homogenous Ligand-Receptor Binding Assay.

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• NanoLuc Binary Technology (NanoBiT) comprises LgBiT and complementation tags (SmBiT, HiBiT).
• NanoBiT has been applied to ligand-binding assays for G protein-coupled receptors (GPCRs).
• This involves genetic fusion of secretory LgBiT (sLgBiT) to GPCRs and SmBiT tagging of peptide ligands.

Purpose of the Study:

• To provide detailed protocols for a NanoBiT-based homogenous ligand-receptor binding assay.
• To establish a novel tool for studying GPCR-ligand interaction mechanisms.
• To demonstrate the assay's utility using the growth hormone secretagogue receptor type 1a (GHSR1a) model system.

Main Methods:

• Genetic fusion of sLgBiT to the N-terminus of GHSR1a.
• Covalent attachment of the SmBiT tag to peptide ligands of GHSR1a.
• Development of a homogenous binding assay protocol utilizing NanoBiT complementation.

Main Results:

• The NanoBiT assay is convenient and suitable for both wild-type and mutant receptors.
• Successful application of NanoBiT for ligand-binding assays of GHSR1a.
• Demonstration of the assay's effectiveness with endogenous agonists and antagonists.

Conclusions:

• NanoBiT provides a novel and convenient homogenous assay for GPCR ligand-binding studies.
• This technology is valuable for investigating receptor-ligand interaction mechanisms.
• Detailed protocols are provided for the GHSR1a model system, facilitating broader application.