Moesin Controls Clathrin-Mediated S1PR1 Internalization in T Cells

The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation.脂质介质鞘氨醇1-磷酸（sphingosine 1-phosphate, S1P）可调控广泛的细胞生理活动，涵盖血管成熟、血管生成以及免疫细胞迁移。目前已鉴定出S1P的5种受体（S1PR1-S1PR5），其中S1PR1是淋巴细胞迁移的关键调控因子：其信号通路是淋巴细胞从淋巴器官迁出的必要条件，而激动剂诱导的受体内吞下调，则是淋巴细胞从血流进入淋巴器官的前提条件。尽管S1PR1下调对淋巴细胞行为的调控具有重要意义，但淋巴细胞中S1PR1内吞的分子机制至今尚未阐明。本研究证实，T细胞内激动剂诱导的S1PR1内吞依赖网格蛋白介导的内吞途径，且受埃兹蛋白-根蛋白-膜突蛋白（ezrin-radixin-moesin, ERM）家族成员膜突蛋白的调控。在经S1P刺激的T细胞中，S1PR1可定位于网格蛋白包被囊泡（clathrin-coated vesicles, CCVs）与早期内体中；若通过药理学手段抑制网格蛋白的功能，则可阻断S1PR1的内吞过程。对膜突蛋白缺陷型T细胞施加S1P刺激，无法诱导S1PR1内吞及网格蛋白包被囊泡的形成。进一步实验显示，使用FTY720——一种已知可介导S1PR1内吞的S1P受体激动剂——处理膜突蛋白缺陷型小鼠时，会引发延迟出现的淋巴细胞减少症；且从经FTY720处理的膜突蛋白缺陷型小鼠体内分离的淋巴细胞，在体外趋化实验中仍可对S1P产生应答。上述结果揭示了膜突蛋白通过调控网格蛋白包被囊泡形成，进而参与网格蛋白依赖型S1PR1内吞调控的全新生物学功能。