Receptor engineering constitutes feedback control and robustness of IL-23R signaling and highlights importance of intracellular cytokine receptor signaling motifs.

BACKGROUND: In the current medical landscape, synthetic cytokine receptors have emerged as a pivotal component in the development of novel therapeutic interventions. Interleukin-23 (IL-23) is the dominant regulatory cytokine in a cluster of immune-mediated inflammatory diseases induced due to the increased expression of the IL-23 receptor (IL-23R) on pathogenic TH17 cells. The modulation of IL-23 signaling by altering IL-23R presents a promising avenue for further investigation. Chimeric cytokine receptors expressing the extracellular domain (ECD) of one protein and the intracellular domain (ICD) of another have been used to isolate the effects of ligand binding from signaling. METHODS: We designed chimeric IL-23Rs that comprise the extracellular and transmembrane domains of IL-23R, as well as various parts of the intracellular region of gp130, the IL-6 signal transducing receptor. To characterize signaling properties by these synthetic cytokine receptors analysis of the activation of the signaling proteins ERK1/2 and STAT3, and SOCS3 was combined with multiplexed single-cell flow cytometry data and information theoretic approaches. RESULTS: Simply transferring the SOCS3-binding site from the IL-6 signal transducing receptor gp130 to IL-23R was not enough to make IL-23 signaling sensitive to SOCS3. However, an iterative transfer process identified a region of gp130 that bound SOCS3 and rendered IL-23 signaling sensitive to SOCS3 regulation. Opposingly, SOCS3-independent gp130 hyper-signaling was achieved by transferring a minimal IL-23R ERK activation motif to gp130. Notably, this motif retained gp130-dependent ERK activation without negative SOCS3 feedback. CONCLUSIONS: In summary, this study identifies the WLYEDIPN motif in IL-23R as an indispensable motif for IL-23R signal transduction and emphasizes the complexity of IL-23-induced signaling pathways and the function of various tyrosines in the intracellular part of cytokine/IL-23 receptor(s).