Visualization and quantification of RANK-RANKL binding for application to disease investigations and drug discovery

Area of Science:

• Biochemistry
• Cell Biology
• Bone Biology

Background:

• Receptor activator of NFκB (RANK) and its ligand (RANKL) binding are crucial for osteoclast differentiation and bone remodeling.
• Imbalances in bone resorption and formation lead to bone diseases like osteopetrosis.
• Understanding RANK-RANKL interactions is vital for developing treatments for bone disorders.

Purpose of the Study:

• To develop a real-time detection method for RANK-RANKL binding using NanoLuc technology.
• To investigate the role of specific RANK and RANKL mutations in autosomal recessive osteopetrosis (ARO).
• To validate the assay's utility in screening compounds that inhibit RANK-RANKL interactions.

Main Methods:

• Co-expression of Large-BiT-RANK and RANKL-Small-BiT fusion proteins in HeLa cells.
• Real-time detection of chemiluminescence upon RANK-RANKL binding.
• Utilizing neutralization antibodies and site-directed mutagenesis to confirm binding specificity.
• Employing a nuclear factor kappa B reporter gene assay to assess signal transduction.

Main Results:

• The NanoLuc assay successfully detected and quantified RANK-RANKL binding in living cells.
• Mutations in RANK or RANKL associated with ARO abolished luminescence and impaired osteoclastogenesis.
• The assay demonstrated inhibition by an anti-RANKL antibody, confirming specificity.
• Impaired signal transduction was observed for the RANK (G54R) mutant.

Conclusions:

• The NanoLuc method provides a robust tool for real-time RANK-RANKL binding detection.
• Loss of RANK-RANKL binding due to specific mutations is a key factor in osteoclast-poor ARO.
• This assay is valuable for mechanistic studies of ARO and for screening anti-osteoporosis drug candidates.