Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET

Interleukin-23 (IL-23) is a pro-inflammatory cytokine involved in the host defense against pathogens but is also implicated in the development of several autoimmune disorders. The IL-23 receptor has become a key target for drug discovery, but the exact mechanism of the receptor ligand interaction remains poorly understood. In this study the affinities of IL-23 for its individual receptor components (IL23R and IL12Rβ1) and the heteromeric complex formed between them have been measured in living cells using NanoLuciferase-tagged full-length proteins. Here, we demonstrate that TAMRA-tagged IL-23 has a greater than 7-fold higher affinity for IL12Rβ1 than IL23R. However, in the presence of both receptor subunits, IL-23 affinity is increased more than three orders of magnitude to 27 pM. Furthermore, we show that IL-23 induces a potent change in the position of the N-terminal domains of the two receptor subunits, consistent with a conformational change in the heteromeric receptor structure. [Display omitted] •NanoBRET assays were created to monitor the interaction of IL-23 with its receptor•The affinities of IL-23 for IL23R, IL12Rβ1 and heteromer complexes were measured•Receptor monomers were found to associate in complexes in the absence of IL-23•IL-23 binding induced a rearrangement of the N-terminal domains of the receptor Lay et al. measure the binding of TAMRA-labeled IL-23 to NanoLuciferase fused receptor subunits, revealing a high-affinity binding site formed from a heteromer of IL23R and IL12Rβ1. NanoBRET was also used to measure the interaction of subunits, demonstrating that IL-23 binding causes a conformational change of pre-formed receptor heteromers.