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。这项工作包括演示参与DAP 12早期信号传导的激酶，描绘所涉及的衔接子，并研究这些途径的调节。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，该系统涉及受体、DAP 12、激酶和报告质粒的转染。该系统允许通过共转染分析途径的各种组分。此外，该途径的生化分析已经确定了DAP 12激活激酶的几种底物。这些底物在介导特异性DAP 12驱动反应中的确切作用正在研究中。此外，对于DAP 12，我们正在开始研究DAP 10，这是已知与NK细胞和单核细胞内的受体相关的第二条链，位于19号染色体上距离DAP 12仅130 bp处。DAP 10含有与DAP 12不同的基于酪氨酸的基序。该基序（Y*xNM）表明与磷脂酰肌醇3激酶（PI 3 K）和衔接子Grb 2两者的相互作用。我们现在正准备剖析DAP 10的信号传导，以充分了解这些链在NK细胞、单核细胞和树突状细胞中的作用。 我们对配对受体系统的研究现在已经很大程度上转移到了对髓样细胞表达的触发受体（TREM）的研究。我们最近鉴定的TLT-1，TREM簇内的一个假定的抑制性受体，定义了TREM作为配对受体系统。TREM-1最近已被证明参与导致败血性休克的信号放大。据报道，TREM-2参与树突状细胞的成熟。总之，这些数据表明TREM参与先天性和适应性免疫应答的调节。TLT-1在TREM的调节中所起的作用（如果有的话）现在正在调查中。TLT-1的表达模式反映了TREM-1的表达模式，并且我们已经证明了TLT-1能够磷酸化并募集蛋白磷酸酶SHP-1。此外，我们已经产生了含有与人IgG的Fc区融合的TLT-1的胞外部分的融合蛋白。使用这种可溶性融合蛋白作为探针，我们已经开始寻找TLT-1配体。我们认为，这些研究将揭示TLT-1在免疫调节中的作用，从而鉴定TLT-1途径中的药物靶点，这些靶点可能在控制病毒感染、自身免疫和恶性肿瘤中很重要。

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.