{"doi":"10.1016/j.jbc.2021.101295","title":"Tryptophan (W) at position 37 of murine IL-12/IL-23 p40 is mandatory for binding to IL-12Rβ1 and subsequent signal transduction","abstract":"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. 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. The interleukin (IL-)12 type cytokines IL-12 and IL-23 play critical roles in a variety of innate and acquired immune responses. Misregulation eventually causes chronic autoimmune responses, and Ustekinumab, an antibody blocking IL-12/IL-23 signaling, was recently approved for psoriasis, psoriatic arthritis, Crohn’s disease, and ulcerative colitis (1Floss D.M. Moll J.M. Scheller J. IL-12 and IL-23-close relatives with structural homologies but distinct immunological functions.Cells. 2020; 9: 2184Crossref Scopus (14) Google Scholar). Knowledge about structure and composition of the IL-12/IL-23 cytokine:recepto","journal":"Journal of Biological Chemistry","year":2021,"id":184630,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":11,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9462,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":740534,"name":"Yvonne Arlt","orcid":null,"position":1,"is_corresponding":false},{"id":739939,"name":"Jens M. Moll","orcid":"0000-0003-3508-5828","position":2,"is_corresponding":false},{"id":739940,"name":"Meryem Ouzin","orcid":"0000-0002-9609-1642","position":3,"is_corresponding":false},{"id":739941,"name":"Hendrik T. Weitz","orcid":"0000-0002-7185-0016","position":4,"is_corresponding":false},{"id":739942,"name":"Lothar Gremer","orcid":"0000-0001-7065-5027","position":5,"is_corresponding":false},{"id":583140,"name":"Dieter Willbold","orcid":"0000-0002-0065-7366","position":6,"is_corresponding":false},{"id":740535,"name":"Joachim Grötzinger","orcid":null,"position":7,"is_corresponding":false},{"id":740536,"name":"Felix Thives-Kurenbach","orcid":null,"position":8,"is_corresponding":false},{"id":739943,"name":"Jürgen Scheller","orcid":"0000-0001-9932-1055","position":9,"is_corresponding":false},{"id":739944,"name":"Doreen M. Floß","orcid":"0000-0002-6675-5313","position":10,"is_corresponding":false},{"id":740533,"name":"Jacqueline Georgy","orcid":null,"position":0,"is_corresponding":true}],"reference_count":28,"raw_metadata":null,"created_at":"2026-07-18T23:48:30.970730Z","pmid":"34637790","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}