Department of Biomedical Sciences, Graduate School of Seoul National University, Seoul 110-799, Korea;
J Immunol. 2013 Dec 1;191(11):5730-42. doi: 10.4049/jimmunol.1203062. Epub 2013 Oct 16.
Mouse CD99 and its paralog CD99-like 2 (CD99L2) are surface proteins implicated in cellular adhesion and migration. Although their distributions overlap in a wide variety of cells, their physical/functional relationship is currently unknown. In this study, we show the interaction between the two molecules and its consequence for membrane trafficking of mouse (m)CD99L2. The interaction was analyzed by bimolecular fluorescence complementation, immunoprecipitation, and fluorescence resonance energy transfer assays. When coexpressed, mCD99 formed heterodimers with mCD99L2, as well as homodimers, and the heterodimers were localized more efficiently at the plasma membrane than were the homodimers. Their interaction was cytoplasmic domain-dependent and enhanced mCD99L2 trafficking to the plasma membrane regardless of whether it was transiently overexpressed or endogenously expressed. Surface levels of endogenous mCD99L2 were markedly low on thymocytes, splenic leukocytes, and CTL lines derived from CD99-deficient mice. Importantly, the surface levels of mCD99L2 on mCD99-deficient cells recovered significantly when wild-type mCD99 was exogenously introduced, but they remained low when a cytoplasmic domain mutant of mCD99 was introduced. Our results demonstrate a novel role for mCD99 in membrane trafficking of mCD99L2, providing useful insights into controlling transendothelial migration of leukocytes.
小鼠 CD99 及其同源物 CD99L2 是参与细胞黏附和迁移的表面蛋白。尽管它们在多种细胞中的分布有重叠，但它们的物理/功能关系目前尚不清楚。在本研究中，我们证明了这两个分子之间的相互作用及其对小鼠（m）CD99L2 膜运输的影响。通过双分子荧光互补、免疫沉淀和荧光共振能量转移实验分析了相互作用。共表达时，mCD99 与 mCD99L2 形成异二聚体以及同二聚体，并且异二聚体比同二聚体更有效地定位于质膜。它们的相互作用依赖于细胞质结构域，并且增强了 mCD99L2 的质膜转运，无论其是否瞬时过表达或内源性表达。在缺乏 CD99 的胸腺细胞、脾白细胞和 CTL 系中，内源性 mCD99L2 的表面水平明显较低。重要的是，当外源性引入野生型 mCD99 时，mCD99 缺陷细胞上 mCD99L2 的表面水平显著恢复，但当引入 mCD99 的细胞质结构域突变体时，其表面水平仍然较低。我们的结果证明了 mCD99 在 mCD99L2 膜运输中的新作用，为控制白细胞跨内皮迁移提供了有用的见解。
