Centre for Cardiovascular Sciences, Institute for Biomedical Research, The College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
Blood. 2010 Apr 8;115(14):2938-46. doi: 10.1182/blood-2009-12-257212. Epub 2010 Feb 12.
The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-beta-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A(2) (TxA(2)). The role of ADP and TxA(2) in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVI-FcRgamma-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation.
C 型凝集素样受体 2（CLEC-2）通过Src 和 Syk 酪氨酸激酶激活血小板，其通过一个称为免疫球蛋白酪氨酸基激活基序（hemITAM）的单一细胞质 YxxL 基序。在这里，我们通过蔗糖梯度超速离心和甲基-β-环糊精处理证明，CLEC-2 在配体结合后向脂筏移位，并且移位对于 hemITAM 磷酸化和信号起始是必需的。hemITAM 磷酸化，但不是移位，也严重依赖于肌动蛋白聚合、Rac1 激活以及 ADP 和血栓烷 A2（TxA2）的释放。ADP 和 TxA2 在介导磷酸化中的作用依赖于配体结合和 rac 激活，但不依赖于血小板聚集。相比之下，具有 2 个 YxxL 基序的 GPVI-FcRgamma-链 ITAM 的酪氨酸磷酸化不依赖于肌动蛋白聚合和二级介质。这些结果揭示了 CLEC-2 磷酸化中涉及肌动蛋白聚合、二级介质和 Rac 激活的一系列独特的近端事件。
