Signal Transduction of Paired Inhibitory Receptors of NK

这个项目涉及一组快速出现的免疫受体的研究。最近在小鼠和人类中发现了许多抑制性免疫受体家族。有趣的是，在这些抑制受体家族中，有一些蛋白质失去了抑制结构域。相反，这些受体在其跨膜区域内获得了带正电的酸，这表明它们可能与信号转导链相互作用并传递正信号。在本项目中，我们研究免疫细胞功能的阳性和阴性调节因子的信号转导和生物化学。通过对阳性受体的研究，我们等人证明了其中一些受体与新型信号转导链DAP12的关联。从那时起，我们一直在表征DAP12信号转导途径的生物化学。我们对配对受体系统的研究目前主要涉及NK细胞的Ly49s和髓系细胞（TREM）上表达的触发受体的研究。在NK细胞中，我们专注于分析各种适配器在DAP12信号传输中的作用。我们已经积累了这些接头的多个敲除小鼠，并在这些小鼠中检测Ly49， NKG2D和Fc受体的功能。杂交不同的菌株将产生多个敲除，以供进一步研究。与NK细胞相比，TREM是一个迅速出现的免疫受体家族。TREM-1最近被证明参与导致感染性休克的信号放大，TREM-2被报道参与树突状细胞的成熟。尽管trem在先天免疫和适应性免疫中都有明确的作用，但其家族的信号转导仍然相对未知。除了TLT-1（见下文）外，每个TREM家族成员都与DAP12信号链物理耦合。然而，由于Fc受体的表达，使用抗体交联TREM的研究很难解释。因此，我们最近开发了一种无抗体的嵌合受体方法来研究髓系室中的DAP12信号传导。该系统利用GPVI的配体结合结构域与DAP12的细胞质结构域的融合。用可溶性配体抽搐素刺激细胞，结果是嵌合体磷酸化，Syk激活，下游信号包括Erk1/2激活。我们现在使用该系统来解剖RAW264巨噬细胞中的DAP12信号。除了TREM家族的激活成员外，我们最近还描述了TREM类转录-1，这是TREM簇中的一种推定的抑制受体。免疫组织化学和免疫荧光研究表明，骨髓TLT-1完全来源于巨核细胞。TLT-1在巨核细胞和血小板中的亚细胞定位和调控表明，TLT-1被巨核细胞预先包装到血小板α颗粒中，以便在血小板脱粒后快速表面表达。这些发现表明TLT-1可能参与α颗粒的产生和释放和/或活化后血小板表面发生的血栓反应。为了充分阐明TLT-1的作用，我们最近采用了细菌重组。这项技术允许在细菌体内重组对大片段DNA进行快速修饰。我们现在已经使用这种技术生成了传统的和bac衍生的TLT-1基因靶向载体。传统构建体的侧翼同源性为6Kb，而基于bac的载体的侧翼同源性接近250kb。将这两种构建体导入C57BL/6 ES细胞，首次直接比较了同源臂长对重组率的影响。我们的结果表明，bac衍生结构的长同源臂产生了5倍的同源重组。两种结构的胚胎干细胞现在都被注射用于生产TLT-1-/-小鼠。这些小鼠将被严格测试TLT-1对血栓形成的影响。当我们开发小鼠模型时，我们正在剖析TLT-1的细胞内结合伙伴。到目前为止，GST下拉和蛋白质测序的结合已经确定了两个合作伙伴，我们正在进一步研究这些合作伙伴。其中一个TLT-1结合蛋白是Nedd4，一个含有E3泛素连接酶的WW结构域。我们目前的模型表明，Nedd4可能参与了TLT-1的包装和/或运输到α颗粒。这些可能性正在使用各种模型来解决。

This project involves the study of a rapidly emerging group of immune receptors. Many families of inhibitory immune receptors have recently been identified in both mice and humans. Interestingly, within each of these inhibitory families of receptors, there are proteins that have lost the inhibitory domains. Instead these receptors have gained a positively charged acid within their transmembrane domain, suggesting that they may interact with signal transduction chains and transmit positive signals. In this project, we study the signal transduction and biochemistry of both the positive and negative regulators of immune cell function. Through the study of the positive receptors, we and others demonstrated the association of some of these receptors with the novel signal transduction chain DAP12. Since then we have been characterizing the biochemistry of the DAP12 signal transduction pathway.Our studies of paired receptor systems have now largely involve the study of the Ly49s of NK cells and the Triggering Receptors Expressed on Myeloid cells (TREM). In NK cells we are focused on dissection of the role of a variety of adaptors in the transmission of the DAP12 signal. We have accumulated multiple knockout mice for these adaptors and are assaying Ly49, NKG2D and Fc receptor function in these mice. Crossing the various strains will generate multiple knockouts for further study. In contrast to NK cells, the TREM are a rapidly emerging immune receptor family. TREM-1 has recently been shown to be involved in the amplification of signals leading to septic shock and TREM-2 has been reported to be involved in the maturation of dendritic cells. Despite the established role of TREMs in both innate and adaptive immunity the signal transduction of the family is still relatively unknown. Each TREM family member, with the exception of TLT-1 (see below), is physically coupled to the DAP12 signaling chain. However, because of the expression of Fc receptors, studies using antibodies to cross link TREM are difficult to interpret. Therefore, we have recently developed an antibody-free, chimeric receptor approach to study DAP12 signaling in the myeloid compartment. The system uses the fusion of the ligand binding domain of GPVI to the cytoplasmic domain of DAP12. Stimulation of the cells with the soluble ligand, convulxin, results is phosphorylation of the chimera, activation of Syk, and downstream signaling including Erk1/2 activation. We are now using this system to dissect DAP12 signaling in RAW264 macrophages.In addition to the activating members of the TREM family, we recently described TREM Like Transcript-1, a putative inhibitory receptor within the TREM cluster. Immunohistochemical and immunofluorescence studies show that bone marrow TLT-1 is derived exclusively from megakaryocytes. The subcellular localization and regulation of TLT-1 in megakaryocytes and platelets suggests that TLT-1 is prepackaged into the platelet alpha granules by megakaryocytes for rapid surface expression after platelet degranulation. These findings suggest TLT-1 may be involved in the production and release of alpha granules and/or the thrombotic response that takes place on the platelet surface after activation. To fully elucidate the role of TLT-1 we have recently employed bacterial recombineering. This technology allows for the rapid modification of large pieces of DNA using in vivo recombination in bacteria. We have now used this technique to generate both traditional and BAC-derived gene targeting vectors for TLT-1. The traditional construct has 6Kb of flanking homology where as the BAC-based vector has nearly 250 Kb of homology. Both constructs were introduced into C57BL/6 ES cells and, for the first time, directly compared the effects of homologous arm length on rates of recombination. Our results showed that the long homologous arms of the BAC-derived construct yielded a five-fold increase in homologous recombination. The ES cells of both constructs are now being injected for the production of TLT-1-/- mice. These mice will be rigorously tested for the effects of TLT-1 on thrombosis.As we develop the mouse model we are dissecting the intracellular binding partners of TLT-1. A combination of GST pulldowns and protein sequencing has identified 2 partners so far and we are investigating these further. One of the TLT-1 binding proteins is Nedd4, a WW domain containing E3 ubiquitin ligase. Our current model suggests that Nedd4 may participate in the packaging and/or trafficking of TLT-1 to the alpha granules. These possibilities are being addressed using a variety of models.