Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction

Interleukin (IL)-12 and IL-23 are composite cytokines consisting of p35/p40 and p19/p40, respectively, which signal via the common IL-12 receptor beta 1 (IL-12R beta 1) and the cytokinespecific receptors IL-12R beta 2 and IL-23R. Previous data showed that the p40 component interacts with IL-12R beta 1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12R beta 2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12R beta 1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL12R beta 2 are known, and two regions of p40 critical for binding to IL-12R beta 1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12R beta 1, we generated deletion variants of the p40-p19 fusion cytokine. We found that an N-terminal deletion variant missing amino acids M23 to P39 failed to induce IL-23 dependent signaling and did not bind to IL-12R beta 1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12R beta 1, but not binding to IL 23R. Combining p40W37K with D36K and T38K mutations eliminated the biological activity of IL-23. Finally, homodimeric p40D36K/W37K/T38K did not interact with IL-12R beta 1, indicating binding of homodimeric p40 to IL-12R beta 1 is comparable to the interaction of IL-23/IL-12 and IL-12R beta 1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL12R beta 1. Structural insights into cytokine-cytokine receptor binding are important to develop novel therapeutic strategies.

Automated summary

This study identified key amino acid residues in the p40 subunit of IL-12/IL-23 that are crucial for binding to IL-12Rβ1, providing insights into the structural basis of cytokine-cytokine receptor interactions and offering potential for the development of novel therapeutic strategies.