Meiser Andrea, Mueller Anja, Wise Emma L, McDonagh Ellen M, Petit Sarah J, Saran Namita, Clark Peter C, Williams Timothy J, Pease James E
Leukocyte Biology Section, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, South Kensington Campus, London, UK.
J Immunol. 2008 May 15;180(10):6713-24. doi: 10.4049/jimmunol.180.10.6713.
The chemokine receptor CXCR3 is expressed on the surface of both resting and activated T lymphocytes. We describe in this study the endocytosis of CXCR3 using T lymphocytes and CXCR3 transfectants. Chemokine-induced CXCR3 down-regulation occurred in a rapid, dose-dependent manner, with CXCL11 the most potent and efficacious ligand. Endocytosis was mediated in part by arrestins, but appeared to occur independently of clathrin and caveolae. In contrast to other chemokine receptors, which are largely recycled to the cell surface within an hour, cell surface replenishment of CXCR3 occurred over several hours and was dependent upon mRNA transcription, de novo protein synthesis, and transport through the endoplasmic reticulum and Golgi. Confocal microscopy and Western blotting confirmed the fate of endocytosed CXCR3 to be degradation, mediated in part by lysosomes and proteosomes. Site-directed mutagenesis of the CXCR3 C terminus revealed that internalization and degradation were independent of phosphorylation, ubiquitination, or a conserved LL motif. CXCR3 was found to be efficiently internalized in the absence of ligand, a process involving a YXXL motif at the extreme of the C terminus. Although freshly isolated T lymphocytes expressed moderate cell surface levels of CXCR3, they were only responsive to CXCL11 with CXCL9 and CXCL10 only having significant activity on activated T lymphocytes. Thus, the activities of CXCR3 are tightly controlled following mRNA translation. Because CXCR3(+) cells are themselves a source of IFN-gamma, which potently induces the expression of CXCR3 ligands, such tight regulation of CXCR3 may serve as a control to avoid the unnecessary amplification of activated T lymphocyte recruitment.
趋化因子受体CXCR3在静止和活化的T淋巴细胞表面均有表达。我们在本研究中描述了利用T淋巴细胞和CXCR3转染细胞对CXCR3的内吞作用。趋化因子诱导的CXCR3下调以快速、剂量依赖性方式发生，其中CXCL11是最有效力和功效的配体。内吞作用部分由抑制蛋白介导，但似乎独立于网格蛋白和小窝发生。与其他趋化因子受体不同，其他趋化因子受体在一小时内大部分会循环回到细胞表面，而CXCR3的细胞表面补充需要数小时，且依赖于mRNA转录、从头蛋白质合成以及通过内质网和高尔基体的运输。共聚焦显微镜和蛋白质免疫印迹证实内化的CXCR3的命运是降解，部分由溶酶体和蛋白酶体介导。对CXCR3 C末端进行定点诱变显示内化和降解与磷酸化、泛素化或保守的LL基序无关。发现CXCR3在没有配体的情况下也能有效内化，这一过程涉及C末端极端处的YXXL基序。尽管新鲜分离的T淋巴细胞表面CXCR３表达水平中等，但它们仅对CXCL11有反应，而CXCL9和CXCL10仅对活化的T淋巴细胞有显著活性。因此，CXCR3的活性在mRNA翻译后受到严格控制。由于CXCR3(+)细胞本身是γ干扰素的来源，γ干扰素能有效诱导CXCR3配体的表达，对CXCR3如此严格的调控可能起到一种控制作用，以避免活化T淋巴细胞募集的不必要扩增。
