MAST CELL INHIBITORY RECEPTORS OF THE GP49 FAMILY

肥大细胞表面FcepsilonR1的交联激活细胞内信号通路，导致生物活性介质的释放，其中许多介导了包括支气管哮喘在内的过敏性疾病的发病机制。gp49家族是免疫球蛋白（Ig）超家族的一个子集，其成员具有明显同源的Ig样结构域，一些分子在其细胞质结构域也具有多个基于酪氨酸的免疫受体抑制基序（ITIMs）。小鼠gp49家族ITIM受体，如gp49Bl和PIR-B与肥大细胞上交联的FcepsilonR1结合，通过依赖于受体与酪氨酸磷酸酶SHP-1的结合，抑制分泌颗粒介质的胞外分泌。本研究的主要目的是研究ITIM受体在小鼠肥大细胞中的表达、功能和信号转导机制。特异性目标1将定义gp49家族ITIM受体在三种表型不同的小鼠肥大细胞群中的表达多样性：通过干细胞因子（SCF）培养的骨髓中获得的固定祖细胞，IL-6和IL-10未成熟肥大细胞，IL-3培养的骨髓中获得的未成熟肥大细胞，以及体外来源的血清肥大细胞，它们是成熟肥大细胞的典型代表。新的gp49家族ITIM受体将通过分子克隆方法和染色体“行走”来寻找，类似于项目1中用于扩大小鼠肥大细胞蛋白酶家族的方法。现有的和任何新的gp49家族ITIM受体的表达差异将在肥大细胞群体的mRNA和蛋白水平上进行监测。特异性Aim 2将比较gp49家族ITIM受体抑制不同肥大细胞群分泌颗粒花生四烯酮衍生和细胞因子介质释放的能力。此外，这些分子的先天抑制能力将通过在大鼠肥大细胞系中表达每个基因家族ITIM受体的细胞质结构域作为具有共同细胞外和跨膜结构域的嵌合体来确定。特异性目的3将阐明gp49家族ITIM受体抑制剂介质释放的机制，重点关注分子胞质域的结构决定因素，以及它们如何与近端抑制酶相互作用，其底物也将被确定。由于拟议的研究旨在增加对肥大细胞活化如何被内源性细胞表面受体反向调节的理解，因此该项目与该应用的中心主题密切相关，即肥大细胞释放介质的调节是过敏性炎症过程中的基本决定因素，包括气道。

The crosslinking of FcepsilonR1 on the surface of mast cells activates intracellular signaling pathways that lead to the lease of bioactive mediators, many of which contribute to the pathogenesis of allergic diseases including bronchial asthma. The gp49 family is a subset of the immunoglobulin (Ig) superfamily whose members have significantly homologous Ig-like domains, some molecules also have multiple immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains. The coligation of mouse gp49 family ITIM receptors, such as gp49Bl and PIR-B to crosslinked FcepsilonR1 on mast cells inhibits the exocytosis of secretory granule mediators in a manner that is dependent on the association of the receptors with the tyrosine phosphatase SHP-1. The broad objective of the proposed research is to address the expression, function, and signal transducing mechanisms of ITIM receptors in mouse mast cells. Specific Aim 1 will define the diversity of gp49 family ITIM receptor expression in three phenotypically distinct mouse mast cell populations: a committed progenitor derived by culture of bone marrow in stem cell factor (SCF), IL-6, and IL-10 immature mast cells obtained from bone marrow cultured in IL-3: and ex vivo-derived serosal mast cells, which typify a mature mast cell. Novel gp49 family ITIM receptors will be sought by molecular cloning approaches and chromosome "walking," analogous to an approach used in Project 1 to expand the family of mouse mast cell proteases. Differences in the expression of existing and any novel gp49 family ITIM receptors will be monitored in the mast cell populations at the mRNA and protein levels. Specific Aim 2 will compare the capacity of gp49 family ITIM receptors to inhibit the release of secretory granule arachidonate-derived, and cytokine mediators from the distinct mast cell populations In addition, the innate inhibitory capacities of the molecules will be determined by expressing the cytoplasmic domain of each gene family ITIM receptor as a chimera with a common extracellular and transmembrane domain in a rat mast cell line. Specific Aim 3 will elucidate the mechanisms by which gp49 family ITIM receptors inhibitor mediator release by focusing on structural determinants in the cytoplasmic domains of the molecules and how they interact with proximal inhibitory enzymes, whose substrates will also be identified. Because the proposed studies are designed to increase understanding of how mast cell activation is counter-regulated by endogenous cell surface receptors, this project is closely related to the central theme of this application, namely, that the regulation of mediator release from mast cells is a fundamental determinant in the course of allergic inflammation, including that of the airways.