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

Jacqueline Georgy, Yvonne Arlt, Jens M Moll, Meryem Ouzin, Hendrik T Weitz, Lothar Gremer, Dieter Willbold, Joachim Grötzinger, Felix Thives-Kurenbach, Jürgen Scheller, Doreen M Floss

The Journal of biological chemistry 2021 Nov

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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 β1 (IL-12Rβ1) and the cytokine-specific receptors IL-12Rβ2 and IL-23R. Previous data showed that the p40 component interacts with IL-12Rβ1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12Rβ2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12Rβ1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL-12Rβ2 are known, and two regions of p40 critical for binding to IL-12Rβ1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12Rβ1, 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-12Rβ1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12Rβ1, 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-12Rβ1, indicating binding of homodimeric p40 to IL-12Rβ1 is comparable to the interaction of IL-23/IL-12 and IL-12Rβ1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL-12Rβ1. Structural insights into cytokine-cytokine receptor binding are important to develop novel therapeutic strategies. Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

Citation

Jacqueline Georgy, Yvonne Arlt, Jens M Moll, Meryem Ouzin, Hendrik T Weitz, Lothar Gremer, Dieter Willbold, Joachim Grötzinger, Felix Thives-Kurenbach, Jürgen Scheller, Doreen M Floss. Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction. The Journal of biological chemistry. 2021 Nov;297(5):101295