MAST CELL INHIBITORY RECEPTORS OF THE GP49 FAMILY

肥大细胞表面FcepsilonR1的交联会激活细胞内信号通路，导致生物活性介质的释放，其中许多生物活性介质参与了包括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，其成员具有显著同源的Ig样结构域，一些分子的胞质结构域中还含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gp49B1和PIR-B，与肥大细胞上的交联FcepsilonR1结合，以依赖于受体与酪氨酸磷酸酶SHP-1的结合的方式抑制分泌颗粒介体的胞吐。本研究的主要目的是探讨ITIM受体在小鼠肥大细胞中的表达、功能和信号转导机制。具体目标1将确定gp49家族ITIM受体在三个不同表型的肥大细胞群中的表达差异：通过干细胞因子(SCF)培养的骨髓衍生的承诺祖细胞，从IL-3培养的骨髓获得的IL-6和IL-10未成熟肥大细胞；以及体外衍生的血清肥大细胞，这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mRNA和蛋白水平上在肥大细胞群体中进行监测。具体目的2将比较gp49家族ITIM受体抑制花生四烯酸分泌颗粒和不同肥大细胞群细胞因子的释放的能力。此外，分子的固有抑制能力将通过将每个基因家族ITIM受体的细胞质结构域表达为具有共同的细胞外和跨膜结构域的嵌合体来确定。具体目标3将通过重点研究gp49家族ITIM受体的胞质区域的结构决定因素以及它们如何与近端抑制酶相互作用来阐明gp49家族ITIM受体抑制物释放的机制，其底物也将被确定。由于拟议的研究旨在增加对肥大细胞激活如何被内源性细胞表面受体反向调节的理解，该项目与本应用的中心主题密切相关，即肥大细胞释放介质的调节是包括呼吸道在内的过敏性炎症过程中的一个基本决定因素。

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.