Murooka Thomas T, Wong Mark M, Rahbar Ramtin, Majchrzak-Kita Beata, Proudfoot Amanda E I, Fish Eleanor N
Division of Cellular and Molecular Biology, Toronto General Research Institute, University Health Network, Department of Immunology, University of Toronto, Toronto, Ontario, M5G 2M1, Canada.
J Biol Chem. 2006 Sep 1;281(35):25184-94. doi: 10.1074/jbc.M603912200. Epub 2006 Jun 28.
CCL5 (RANTES (regulated on activation normal T cell expressed and secreted)) and its cognate receptor, CCR5, have been implicated in T cell activation. CCL5 binding to glycosaminoglycans (GAGs) on the cell surface or in extracellular matrix sequesters CCL5, thereby immobilizing CCL5 to provide the directional signal. In two CCR5-expressing human T cell lines, PM1.CCR5 and MOLT4.CCR5, and in human peripheral blood-derived T cells, micromolar concentrations of CCL5 induce apoptosis. CCL5-induced cell death involves the cytosolic release of cytochrome c, the activation of caspase-9 and caspase-3, and poly(ADP-ribose) polymerase cleavage. CCL5-induced apoptosis is CCR5-dependent, since native PM1 and MOLT4 cells lacking CCR5 expression are resistant to CCL5-induced cell death. Furthermore, we implicate tyrosine 339 as a critical residue involved in CCL5-induced apoptosis, since PM1 cells expressing a tyrosine mutant receptor, CCR5Y339F, do not undergo apoptosis. We show that CCL5-CCR5-mediated apoptosis is dependent on cell surface GAG binding. The addition of exogenous heparin and chondroitin sulfate and GAG digestion from the cell surface protect cells from apoptosis. Moreover, the non-GAG binding variant, (44AANA47)-CCL5, fails to induce apoptosis. To address the role of aggregation in CCL5-mediated apoptosis, nonaggregating CCL5 mutant E66S, which forms dimers, and E26A, which form tetramers at micromolar concentrations, were utilized. Unlike native CCL5, the E66S mutant fails to induce apoptosis, suggesting that tetramers are the minimal higher ordered CCL5 aggregates required for CCL5-induced apoptosis. Viewed altogether, these data suggest that CCL5-GAG binding and CCL5 aggregation are important for CCL5 activity in T cells, specifically in the context of CCR5-mediated apoptosis.
CCL5（调节激活正常T细胞表达和分泌因子（RANTES））及其同源受体CCR5与T细胞激活有关。CCL5与细胞表面或细胞外基质中的糖胺聚糖（GAGs）结合会隔离CCL5，从而固定CCL5以提供定向信号。在两种表达CCR5的人T细胞系PM1.CCR5和MOLT4.CCR5以及人外周血来源的T细胞中，微摩尔浓度的CCL5可诱导细胞凋亡。CCL5诱导的细胞死亡涉及细胞色素c的胞质释放、caspase-9和caspase-3的激活以及聚（ADP-核糖）聚合酶的裂解。CCL5诱导的细胞凋亡是CCR5依赖性的，因为缺乏CCR5表达的天然PM1和MOLT4细胞对CCL5诱导的细胞死亡具有抗性。此外，我们认为酪氨酸339是参与CCL5诱导细胞凋亡的关键残基，因为表达酪氨酸突变受体CCR5Y339F的PM1细胞不会发生凋亡。我们表明，CCL5-CCR5介导的细胞凋亡依赖于细胞表面GAG结合。添加外源性肝素和硫酸软骨素以及从细胞表面消化GAG可保护细胞免于凋亡。此外，非GAG结合变体（44AANA47）-CCL5不能诱导细胞凋亡。为了研究聚集在CCL5介导的细胞凋亡中的作用，使用了微摩尔浓度下形成二聚体的非聚集CCL5突变体E66S和形成四聚体的E26A。与天然CCL5不同，E66S突变体不能诱导细胞凋亡，这表明四聚体是CCL5诱导细胞凋亡所需的最小高阶CCL5聚集体。总体而言，这些数据表明CCL5-GAG结合和CCL5聚集对于T细胞中CCL5的活性很重要，特别是在CCR5介导的细胞凋亡的背景下。
