白细胞介素-31（IL-31）是参与皮肤炎症和肿瘤进展的促炎细胞因子。IL-31信号传导级联是由其与两个受体结合引发的，分别是IL-31受体α（IL-31RA）和生长抑素M受体亚单位β（OSMRβ）。此前的研究表明，人IL-31（hIL-31）能够独立地与IL-31RA和OSMRβ发生直接相互作用，但其与IL-31RA的结合能力要强于OSMRβ。与其人类同源物不同，猫IL-31（fIL-31）对猫OSMRβ的结合亲和性更高。然而，狗IL-31与其受体的结合模式尚未阐明。本研究中，我们纯化了重组犬IL-31（rcIL-31）蛋白，并揭示其二级结构主要由α螺旋组成。此外，体外研究表明，rcIL-31具有诱导DH-82细胞中信号转导激活因子3（STAT3）和STAT5磷酸化的能力。接下来，我们使用流式细胞仪测定了生物活性rcIL-31对其单独受体组分的结合效力。结果表明，正确折叠的rcIL-31能够独立地与表达在细胞表面上的cIL-31RA和cOSMRβ结合。值得注意的是，rcIL-31对OSMRβ的亲和力比对IL-31RA高出十倍以上。此外，我们证明了cOSMRβ的D1-D4，尤其是D4，对其与cIL-31的结合至关重要。此外，本研究证明，rcIL-31对可溶性cOSMRβ具有较高的结合亲和力，KD值为3.59×10-8 M。本研究所呈现的结果对于开发针对cIL-31信号引起的疾病的药物或抗体具有重要意义。© King Abdulaziz City for Science and Technology 2023.Springer Nature或其许可方（例如一个协会或其他合作方）根据与作者或其他权利所有者的一项出版协议独家拥有本文章的全部权利；接受的原始手稿版本的作者自我存档完全受制于该等出版协议及适用法律。

Interleukin-31 (IL-31) is a pro-inflammatory cytokine involved in skin inflammation and tumor progression. The IL-31 signaling cascade is initiated by its binding to two receptors, IL-31 receptor alpha (IL-31RA) and oncostatin M receptor subunit beta (OSMRβ). The previous study suggested that human IL-31 (hIL-31) directly interacts with IL-31RA and OSMRβ, independently, but the binding ability of hIL-31 to IL-31RA is stronger than to OSMRβ. In different to its human ortholog, feline IL-31 (fIL-31) has a higher binding affinity for feline OSMRβ. However, the binding pattern of canine IL-31 to its receptors remains to be elucidated. In this study, we purified the recombinant canine IL-31 (rcIL-31) protein and revealed its secondary structure to be mainly composed of alpha-helices. Moreover, in vitro studies show that rcIL-31 has the ability to induce the phosphorylation of signal transducer activator of transcription 3 (STAT3) and STAT5 in DH-82 cells. In the following, the binding efficacies of bioactive rcIL-31 for its individual receptor components have been measured using a flow cytometry assay. The result demonstrates that correctly refolded rcIL-31 binds independently with cIL-31RA and cOSMRβ which were expressed on the cell surface. Of note, rcIL-31 has a greater than tenfold higher affinity to OSMRβ than to IL-31RA. Additionally, we demonstrated that D1-D4, especially D4 of cOSMRβ, is crucial for its binding to cIL-31. Furthermore, this study proved that rcIL-31 has a high binding affinity to the soluble cOSMRβ with a KD value of 3.59 × 10-8 M. The results presented in the current study will have a significant implication in the development of drugs or antibodies against diseases induced by cIL-31 signaling.© King Abdulaziz City for Science and Technology 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.