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质在许多炎症和免疫反应中发挥重要作用。我们研究的目标是了解导致这些分子释放的细胞内信号转导途径。在之前的研究中，我们证明蛋白酪氨酸激酶 Syk 对于免疫受体诱导的脱颗粒至关重要，从而导致炎症介质的释放。还鉴定出了未检测到 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个 SH2 和激酶结构域之间的 Syk 接头区域，对于调节该激酶的功能非常重要。为了鉴定对信号转导至关重要的蛋白酪氨酸激酶 Lyn 和 Syk 的新底物，我们筛选了由 RBL-2H3 细胞制备的 cDNA 表达文库，以寻找体外酪氨酸磷酸化的蛋白。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。实验正在继续阐明我们发现的几种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，野生型或催化失活形式的 PLD1 或 PLD2 在 RBL-2H3 细胞中稳定过表达。这种受体诱导的 PLD 激活需要蛋白酪氨酸激酶 Syk，并导致 PLD1 和 PLD2 的激活。然而，PLD1 是大多数受体诱导的 PLD 活性的来源。仅在过表达催化失活的 PLD1 的细胞中，受体诱导的脱粒作用增强。研究发现，这与调节磷脂酸形成的基本 PLD1 活性相关，然后控制由免疫受体聚集引发的早期信号。 Cbl 家族蛋白负向调节来自酪氨酸激酶偶联受体的信号传导。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，从野生型、c-Cbl 和 Cbl-b 缺陷小鼠中产生肥大细胞培养物。与对照细胞相比，Cbl-b 失活导致免疫球蛋白受体诱导的信号传导和炎症介质释放增加。与 Cbl-b 相比，c-Cbl 缺乏对受体诱导的脱颗粒或信号转导没有可检测到的影响。这些结果表明，Cbl-b 和 c-Cbl 对免疫受体信号转导具有不同的作用，并且 Cbl-b 而不是 c-Cbl 充当脱颗粒的负调节剂。长期以来，人们一直认为骨髓中存在定型肥大细胞前体，尽管其识别和分离已被证明很困难。我们使用两种肥大细胞特异性抗体进行连续免疫磁分离，以纯化和表征来自成年小鼠骨髓的谱系定型肥大细胞前体，该前体仅占细胞总数的 0.02%。我们研究了这些细胞的体外生长要求和形态，现在正在表征在未成熟前体细胞中独特表达的分子。