Profiling membrane proteins’ interacting networks is crucial for understanding their regulatory mechanisms and functional characteristics, but it remains a challenging task. Here, by combining genetic incorporation of crosslinkers, tandem denatured purification, and proteomics, we added interaction partners for PD-L1, a cancer cell surface protein that inhibits T cell activity. The site-specifically incorporated crosslinker mediates the covalent capture of interactions under physiological conditions and enabled the PD-L1 complexes to withstand the harsh extraction conditions of membrane proteins. Subsequent experiments led to the identification of potential PD-L1 interaction candidates and verified membrane-associated progesterone receptor component 1 as a novel PD-L1 interaction partner in mammalian cells. Importantly, we demonstrated that PGRMC1 positively regulates PD-L1 expression by regulating GSK3β-mediated PD-L1 degradation in cancer cells. Furthermore, PGRMC1 knockdown results in dramatically enhanced T cell-mediated cytotoxicity in cancer cells. In conclusion, our study elucidated the interactome of PD-L1 and uncovered a new player in the PD-L1 regulation mechanism.

分析膜蛋白的相互作用网络对于理解其调节机制和功能特征至关重要，但这仍然是一项具有挑战性的任务。在这里，通过结合交联剂的基因掺入、串联变性纯化和蛋白质组学，我们添加了 PD-L1（一种抑制 T 细胞活性的癌细胞表面蛋白）的相互作用伙伴。位点特异性掺入的交联剂介导生理条件下相互作用的共价捕获，并使 PD-L1 复合物能够承受膜蛋白的严酷提取条件。随后的实验确定了潜在的 PD-L1 相互作用候选者，并验证了膜相关孕酮受体成分 1 作为哺乳动物细胞中新型 PD-L1 相互作用伴侣。重要的是，我们证明 PGRMC1 通过调节癌细胞中 GSK3β 介导的 PD-L1 降解来正向调节 PD-L1 表达。此外，PGRMC1 敲低导致癌细胞中 T 细胞介导的细胞毒性显着增强。总之，我们的研究阐明了 PD-L1 的相互作用组，并发现了 PD-L1 调节机制中的一个新参与者。