Eph（人肝细胞产生红细胞生成素的）受体家族是受体酪氨酸激酶（RTKs）中最大的亚类，对胚胎发育和神经发生起着重要作用。胞内的无菌α结构域（SAM）是区分Eph受体与其他RTKs的关键结构特征，参与招募和结合下游分子。本研究通过SAM-SAM结构域相互作用，确定了SASH1（含有SAM和SH3结构域的1）作为新的Eph受体结合伴侣。我们的全面生化分析表明，SASH1通过其SAM1结构域选择性地与Eph受体相互作用，其中对EphA8的亲和力最高。EphA8-SASH1复合物的高分辨率晶体结构为这些蛋白质之间的特异互作提供了见解。细胞实验验证了EphA8和SASH1在哺乳动物细胞中的共定位和共沉淀，癌变突变体（EphA8 R942H或G978D）削弱了这种相互作用。我们证明了SAM-SAM相互作用对于SASH1介导的EphA8激酶活性调节至关重要，为Eph信号通路提供了新的启示，并扩展了我们对肿瘤抑制基因SASH1分子基础的理解。版权所有 © 2023. 由Elsevier Ltd.出版。

The Eph (erythropoietin-producing human hepatocellular) receptor family, the largest subclass of receptor tyrosine kinases (RTKs), plays essential roles in embryonic development and neurogenesis. The intracellular Sterile Alpha Motif (SAM) domain presents a critical structural feature that distinguishes Eph receptors from other RTKs and participates in recruiting and binding downstream molecules. This study identified SASH1 (SAM and SH3 domain containing 1) as a novel Eph receptor-binding partner through SAM-SAM domain interactions. Our comprehensive biochemical analyses revealed that SASH1 selectively interacts with Eph receptors via its SAM1 domain, displaying the highest affinity for EphA8. The high-resolution crystal structure of the EphA8-SASH1 complex provided insights into the specific intermolecular interactions between these proteins. Cellular assays confirmed that EphA8 and SASH1 co-localize and co-precipitate in mammalian cells, with cancer mutations (EphA8 R942H or G978D) impairing this interaction. We demonstrated that SAM-SAM interaction is critical for SASH1-mediated regulation of EphA8 kinase activity, shedding new light on the Eph signaling pathway and expanding our understanding of the molecular basis of the tumor suppressor gene SASH1.Copyright © 2023. Published by Elsevier Ltd.