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

Charles S Lay,Angela Bridges,Joelle Goulding,Stephen J Briddon,Zoja Soloviev,Peter D Craggs,Stephen J Hill

doi:10.1016/j.chembiol.2021.05.002

Charles S Lay, Angela Bridges + Show 5 more

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https://doi.org/10.1016/j.chembiol.2021.05.002

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Abstract

SummaryInterleukin-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.

Highlights

Interleukin-23 (IL-23) is an important pro-inflammatory cytokine, produced by antigen-presenting cells, such as dendritic cells and macrophages, that plays a crucial role in host defense against bacterial and fungal pathogens (Verreck et al, 2004; Werner et al, 2011)
The specific receptor for the IL-23 cytokine is formed of the IL-12 receptor subunit b1 (IL12Rb1) and the IL-23 receptor (IL23R), which are both needed for subsequent Janus kinase (JAK) activation and signaling (Parham et al, 2002)
Following removal of the unreacted labeling reagent using a desalting column, the TAMRA-conjugated IL-23 and the unlabeled IL-23 were assessed using liquid chromatography coupled with mass spectrometry (LC-MS; Figures S1 and S2)

Summary

Introduction

Interleukin-23 (IL-23) is an important pro-inflammatory cytokine, produced by antigen-presenting cells, such as dendritic cells and macrophages, that plays a crucial role in host defense against bacterial and fungal pathogens (Verreck et al, 2004; Werner et al, 2011). Each IL-12 heteromeric receptor component is thought to have affinity for a specific a or b subunit of each heterodimeric IL-12 family cytokine (Hasegawa et al, 2016). The specific receptor for the IL-23 cytokine is formed of the IL-12 receptor subunit b1 (IL12Rb1) and the IL-23 receptor (IL23R), which are both needed for subsequent Janus kinase (JAK) activation and signaling (Parham et al, 2002). The formation of heteromers by IL-12 family cytokine receptors facilitates promiscuous pairing within the IL-12 family, leading to a diverse range of functional effects (Floss et al, 2020; Hasegawa et al, 2016). IL-23 and IL-12 mediate distinct pathways, with IL-23 inducing the expansion and maintenance of Th17 cells and IL-12, leading to differentiation of Th1 cells (Vignali and Kuchroo, 2012)

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