Use of NanoBiT and NanoBRET to characterise interleukin-23 receptor dimer formation in living cells

Lay, Charles S.; Kilpatrick, Laura E.; Craggs, Peter D.; Hill, Stephen J.

Abstract

Background and Purpose: Interleukin-23 (IL-23) and its receptor are important drug targets for the treatment of auto-inflammatory diseases. IL-23 binds to a receptor complex composed of two single transmembrane spanning proteins IL23R and IL12Rβ1. In this study, we aimed to gain further understanding of how ligand binding induces signalling of IL-23 receptor complexes using the proximity-based techniques of NanoLuc Binary Technology (NanoBiT) and Bioluminescence Resonance Energy Transfer (BRET). Experimental Approach: To monitor the formation of IL-23 receptor complexes, we developed a split luciferase (NanoBiT) assay whereby heteromerisation of receptor subunits can be measured through luminescence. The affinity of NanoBiT complemented complexes for IL-23 was measured using NanoBRET, and cytokine-induced signal transduction was measured using a phospho-STAT3 AlphaLISA assay. Key Results: NanoBiT measurements demonstrated that IL-23 receptor complexes formed to an equal degree in the presence and absence of ligand. NanoBRET measurements confirmed that these complexes bound IL-23 with a picomolar binding affinity. Measurement of STAT3 phosphorylation demonstrated that pre-formed IL-23 receptor complexes induced signalling following ligand binding. It was also demonstrated that synthetic ligand-independent signalling could be induced by high affinity (HiBit) but not low affinity (SmBit) NanoBiT crosslinking of the receptor N-terminal domains. Conclusions and Implications: These results indicate that receptor complexes form prior to ligand binding and are not sufficient to induce signalling alone. Our findings indicate that IL-23 induces a conformational change in heteromeric receptor complexes, to enable signal transduction. These observations have direct implications for drug discovery efforts to target the IL-23 receptor.

Citation

Lay, C. S., Kilpatrick, L. E., Craggs, P. D., & Hill, S. J. (2022). Use of NanoBiT and NanoBRET to characterise interleukin-23 receptor dimer formation in living cells. British Journal of Pharmacology, https://doi.org/10.1111/bph.16018

• Journal Article Type: Article
• Acceptance Date: Dec 19, 2022
• Online Publication Date: Dec 22, 2022
• Publication Date: Dec 22, 2022
• Deposit Date: Jan 4, 2023
• Publicly Available Date: Dec 23, 2023
• Journal: British Journal of Pharmacology
• Print ISSN: 0007-1188
• Electronic ISSN: 1476-5381
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• DOI: https://doi.org/10.1111/bph.16018
• Keywords: NanoBiT, NanoBRET, IL23R, cytokine-receptor, Interleukin-23, IL12Rβ1, STAT3
• Public URL: https://nottingham-repository.worktribe.com/output/15436629
• Publisher URL: https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.16018
• Additional Information: This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.