Signal Transduction of Paired Inhibitory Receptors of NK

该项目涉及对一组迅速出现的免疫受体的研究。许多家族的抑制性免疫受体最近已被确定在小鼠和人类。有趣的是，在这些抑制性受体家族中的每一个中，都有失去抑制性结构域的蛋白质。相反，这些受体在其跨膜结构域中获得了带正电荷的酸，这表明它们可能与信号转导链相互作用并传递正信号。在这个项目中，我们研究了免疫细胞功能的正负调节因子的信号转导和生物化学。通过对阳性受体的研究，我们和其他人证明了这些受体中的一些与新的信号转导链DAP 12的关联。从那时起，我们一直在表征DAP 12信号转导通路的生物化学。我们对配对受体系统的研究现在主要涉及NK细胞的Ly 49和髓样细胞表达的触发受体（TREM）的研究。在NK细胞中，我们专注于解剖各种衔接子在DAP 12信号传递中的作用。我们已经为这些衔接子积累了多个敲除小鼠，并正在分析这些小鼠中的Ly 49，NKG 2D和Fc受体功能。将各种菌株杂交将产生多个敲除用于进一步研究。与NK细胞相比，TREM是一个迅速出现的免疫受体家族。最近已经显示TREM-1参与导致败血性休克的信号的放大，并且已经报道TREM-2参与树突细胞的成熟。尽管TREM在先天性和适应性免疫中的作用已经确定，但该家族的信号转导仍然相对未知。除了TLT-1（见下文）之外，每个TREM家族成员都与DAP 12信号链物理偶联。然而，由于Fc受体的表达，使用抗体交联TREM的研究难以解释。因此，我们最近开发了一种无抗体的嵌合受体方法来研究骨髓室中的DAP 12信号传导。该系统使用GPVI的配体结合结构域与DAP 12的胞质结构域的融合。用可溶性配体惊厥素刺激细胞，导致嵌合体的磷酸化、Syk的活化和下游信号传导，包括Erk 1/2活化。除了TREM家族的激活成员外，我们最近还描述了TREM样转录物-1，一种TREM簇内的假定抑制性受体。免疫组织化学和免疫荧光研究表明，骨髓TLT-1是专门来自巨核细胞。TLT-1在巨核细胞和血小板中的亚细胞定位和调节表明，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形成反应。为了充分阐明TLT-1的作用，我们最近采用了细菌重组工程。该技术允许使用细菌中的体内重组来快速修饰大片段DNA。我们现在已经使用这种技术来产生TLT-1的传统和BAC衍生的基因靶向载体。传统构建体具有6 Kb的侧翼同源性，而基于BAC的载体具有近250 Kb的同源性。这两种构建体被引入C57 BL/6 ES细胞，并首次直接比较了同源臂长度对重组率的影响。我们的结果表明，BAC衍生的构建体的长同源臂在同源重组中产生了五倍的增加。两种构建体的ES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，我们正在解剖TLT-1的细胞内结合伴侣。到目前为止，GST下拉和蛋白质测序的组合已经确定了2个合作伙伴，我们正在进一步调查这些合作伙伴。TLT-1结合蛋白之一是Nedd 4，一种含有E3泛素连接酶的WW结构域。我们目前的模型表明，Nedd 4可能参与包装和/或运输TLT-1的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.