Signal Transduction In Mast Cells

肥大细胞中的信号转导

• 批准号: 6966504
• 负责人: Reuben P. Siraganian
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6966504
• 关键词: basophils; biological signal transduction; immunoglobulins; immunologic receptors; immunomagnetic separation; mast cell; phospholipase D; protein tyrosine kinase

Mast cells play an important role in many inflammatory and immunological reactions by releasing an array of mediators. The goal of our studies is to understand the intracellular signal transduction pathways that lead to the release of these molecules. In previous studies, we demonstrated that the protein tyrosine kinase Syk is essential for the immune receptor-induced degranulation that results in the release of inflammatory mediators. A variant of the rat basophilic RBL-2H3 mast cells that has no detectable Syk was also identified and has been used to examine the structural basis of the regulation of Syk after immune receptor aggregation. These studies identified the linker region of Syk, located between the second SH2 and the kinase domain, as important in regulating the function of this kinase. To identify novel substrates of the protein tyrosine kinases Lyn and Syk that are critical in signaling, we screened a cDNA expression library prepared from RBL-2H3 cells for proteins that were tyrosine phosphorylated in vitro. Five clones as potential Lyn substrates and eight clones as Syk substrates were identified including known molecules. Experiments are continuing to elucidate the functional importance of several new molecules that we identified. To investigate the role of phospholipase D (PLD) in immune receptor signaling, the wild type or the catalytically inactive forms of PLD1 or PLD2 were stably overexpressed in RBL-2H3 cells. This receptor-induced PLD activation required the protein tyrosine kinase Syk and resulted in the activation of both PLD1 and PLD2. However, PLD1 was the source of most of the receptor-induced PLD activity. There was enhanced receptor-induced degranulation only in cells that overexpressed the catalytically inactive PLD1. This was found to correlate with the constitutive basal PLD1 activity that regulates phosphatidic acid formation that then controls the early signals initiated by immune receptor aggregation. The Cbl family proteins negatively regulate signaling from tyrosine kinase-coupled receptors. To examine the role of c-Cbl and Cbl-b in immunoglobulin receptor signaling, mast cell cultures were generated from wild-type, c-Cbl and Cbl-b deficient mice. Compared to control cells, Cbl-b inactivation resulted in increases in immunoglobulin receptor-induced signaling and release of inflammatory mediators. In contrast to Cbl-b, c-Cbl deficiency had no detectable effect on receptor-induced degranulation or signal transduction. These results indicate that Cbl-b and c-Cbl have divergent effects on immune receptor signal transduction and that Cbl-b, but not c-Cbl, functions as a negative regulator of degranulation. A committed mast cell precursor has long been assumed to be present in bone marrow, although its identification and isolation has proven to be difficult. We have used sequential immunomagnetic isolation with two mast cell specific antibodies to purify and characterize a lineage committed mast cell precursor from adult mouse bone marrow that represents only 0.02% of the total cells. We have studied the in vitro growth requirements and morphology of these cells and we are now characterizing the molecules that are uniquely expressed in the immature precursors.

肥大细胞通过释放一系列介体来在许多炎症和免疫反应中起重要作用。我们研究的目的是了解导致这些分子释放的细胞内信号转导途径。在先前的研究中，我们证明蛋白酪氨酸激酶SYK对于免疫受体诱导的脱粒至关重要，从而导致炎症介质的释放。还鉴定了未检测到的SYK的大鼠嗜碱性RBL-2H3肥大细胞的变体，并已用于检查免疫受体聚集后SYK调节的结构基础。这些研究确定了位于第二SH2和激酶结构域之间的SYK的接头区域，对于调节该激酶的功能很重要。为了鉴定蛋白质酪氨酸激酶Lyn和Syk的新型底物在信号传导中至关重要，我们筛选了从RBL-2H3细胞制备的cDNA表达文库，用于在体外磷酸化的酪氨酸磷酸化的蛋白质。鉴定出五个克隆作为潜在的LYN底物和八个克隆作为SYK底物，包括已知分子。实验继续阐明我们确定的几个新分子的功能重要性。为了研究磷脂酶D（PLD）在免疫受体信号传导中的作用，在RBL-2H3细胞中稳定表达了PLD1或PLD2的野生型或催化性不活跃形式。该受体诱导的PLD激活需要蛋白酪氨酸激酶SYK，并导致PLD1和PLD2的激活。但是，PLD1是大多数受体诱导的PLD活性的来源。仅在过表达催化无效PLD1的细胞中，受体诱导的脱粒作用增强。发现这与调节磷脂酸的形成的本构基底PLD1活性相关，然后控制免疫受体聚集引发的早期信号。 CBL家族蛋白对酪氨酸激酶偶联受体的信号传导负调节。为了检查C-CBL和CBL-B在免疫球蛋白受体信号传导中的作用，由野生型，C-CBL和CBL-B缺乏小鼠产生肥大细胞培养物。与对照细胞相比，CBL-B灭活导致免疫球蛋白受体诱导的信号传导和炎症介质的释放增加。与CBL-B相反，C-CBL缺乏对受体诱导的脱粒或信号转导没有可检测的影响。这些结果表明，CBL-B和C-CBL对免疫受体信号转导的影响有分歧，而CBL-B（而不是C-CBL）则是脱粒的负调节剂。长期以来，人们长期以来一直认为施加的肥大细胞前体存在于骨髓中，尽管事实证明其鉴定和隔离很困难。我们已经使用两种肥大细胞特异性抗体的顺序免疫磁分离来纯化和表征来自成年小鼠骨髓的谱系肥大细胞前体，该肥大细胞仅占总细胞的0.02％。我们已经研究了这些细胞的体外生长需求和形态，现在我们正在表征未成熟前体中唯一表达的分子。