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。最近在老鼠和人类身上都发现了许多抑制性免疫受体家族。有趣的是，在这些受体的每个抑制家族中，都有一些蛋白质失去了抑制结构域。相反，这些受体在其跨膜结构域中获得了一个带正电荷的酸，这表明它们可能与信号转导链相互作用，传递积极的信号。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其中一些受体与新的信号转导链DAP12的关联。从那时起，我们一直在研究DAP12信号转导通路的生化特征。这项工作包括展示参与DAP12早期信号传递的激酶，描述所涉及的接头，以及研究这些通路的调节。为了促进这些研究，我们已经开发并正在利用一种重组系统，涉及到受体DAP12、一种激酶和一种报告质粒的转染。该系统允许通过共转染来分析该途径的各种成分。此外，对该途径的生化分析还确定了DAP12激活的几种底物。这些底物在介导特定的DAP12驱动的反应中的确切作用正在研究中。此外，对于DAP12，我们正在开始DAP10的研究，这是一条已知与NK细胞和单核细胞内的受体相关的第二链，位于19号染色体上DAP12的130bp处。DAP10包含一个不同于DAP12的基于酪氨酸的基序。这个基序(Y*xNM)提示与磷脂酰肌醇3激酶(PI3K)和接头Grb2相互作用。我们现在正准备剖析DAP10的信号转导机制，以期全面了解这些链在NK细胞、单核细胞和树突状细胞中的作用。 我们对成对受体系统的研究现在主要转移到对髓样细胞上表达的触发受体(TREM)的研究上。我们最近发现了TLT-1，它是TREM簇中的一种假定的抑制性受体，它将TREM定义为配对受体系统。最近发现TREM-1参与了导致感染性休克的信号放大。据报道，TREM-2参与了树突状细胞的成熟。总而言之，这些数据表明，Trem参与了先天性和获得性免疫反应的调节。TLT-1在Trem的调节中扮演了什么角色，如果有的话，目前正在调查中。TLT-1的表达模式反映了TREM-1的表达模式，我们已经证明了TLT-1被磷酸化并招募蛋白磷酸酶SHP-1的能力。此外，我们还制备了含有TLT-1胞外部分与人免疫球蛋白Fc区融合的融合蛋白。利用该可溶性融合蛋白作为探针，我们已经开始寻找TLT-1的配体。我们认为，这些研究将揭示TLT-1在免疫调节中的作用，从而识别TLT-1途径中可能在控制病毒感染、自身免疫和恶性肿瘤方面重要的药物靶点。

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 the DAP12 signal transduction pathway. This work has included demonstration of the kinases involved in the early signaling of DAP12, delineation of the adaptors involved, and study of the regulation of these pathways. To facilitate these studies, we have developed and are utilizing, a reconstitution system that involves transfection of the receptor, DAP12, a kinase, and a reporter plasmid. This system allows for the analysis of various components of the pathway via co-transfection. In addition, biochemical analysis of the pathway has defined several substrates of DAP12-activated kinases. The exact roles of these substrates in mediating specific DAP12-driven responses is under investigation. In addition, to DAP12, we are beginning the study of DAP10, a second chain known to associate with receptors within NK cells and monocytes that is located just 130 bp from DAP12 on Chromosome 19. DAP10 contains a tyrosine based motif unique from that of DAP12. This motif (Y*xNM) suggests interaction with both the phosphatidylinositol 3 kinase (PI3K) and the adaptor Grb2. We are now preparing to dissect the signaling of DAP10 in an effort to fully understand the role of these chains within NK cells, monocytes and dendritic cells. Our studies of paired receptor systems has now largely shifted to the study of the Triggering Receptors Expressed on Myeloid cells (TREM). Our recent identification of TLT-1, a putative inhibitory receptor within the TREM cluster has defined the TREM as paired receptor system. TREM-1 has recently been shown to be involved in the amplification of signals leading to septic shock. TREM-2 has been reported to be involved in the maturation of dendritic cells. Together these data suggest the TREM are involved in the regulation of both the innate and adaptive immune response. What role, if any, TLT-1 plays in the regulation of the TREM is now under investigation. The pattern of expression of TLT-1 mirrors that of TREM-1, and we have demonstrated the ability of TLT-1 to become phosphorylated and recruit the protein phosphatase SHP-1. In addition, we have produced a fusion protein containing the extracellular portion of TLT-1 fused to the Fc region of human IgG. Using this soluble fusion protein as a probe we have begun to search for TLT-1 ligands. It is our feeling that these studies will uncover the role of TLT-1 in immune regulation allowing for the identification of drug targets within the TLT-1 pathway that may be important in the control of viral infection, autoimmunity, and malignancy.