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

Autor: Charles S. Lay, Laura E. Kilpatrick, Peter D. Craggs, Stephen J. Hill
Rok vydání: 2023
Předmět:
Zdroj: British Journal of Pharmacology. 180:1444-1459
ISSN: 1476-5381
0007-1188
Popis: 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).To monitor the formation of IL-23 receptor complexes, we developed a split luciferase (NanoBiT) assay whereby heteromerization 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.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.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.
Databáze: OpenAIRE
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