Signal Transduction In Mast Cells

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。在以前的研究中，我们证明了蛋白酪氨酸激酶Syk在免疫受体诱导的脱颗粒过程中是必不可少的，这种脱颗粒导致炎症介质的释放。还发现了大鼠嗜碱性RBL-2H3肥大细胞的一个变体，它没有检测到Syk，并已用于研究免疫受体聚集后Syk调节的结构基础。这些研究发现，Syk的连接区位于第二个SH2和激酶结构域之间，在调节该激酶的功能方面起着重要的作用。为了确定在信号转导中起关键作用的蛋白酪氨酸激酶Lyn和Syk的新底物，我们在体外筛选了从RBL-2H3细胞中制备的酪氨酸磷酸化蛋白的cDNA表达文库。鉴定出5个克隆为潜在的Lyn底物，8个克隆为Syk底物，其中包括已知分子。实验正在继续阐明我们确定的几个新分子的功能重要性。为探讨磷脂酶D(PLD)在免疫受体信号转导中的作用，在RBL-2H3细胞中稳定过表达野生型或非催化活性PLD1或PLD2。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。只有在过度表达催化失活的PLD1的细胞中，才有增强的受体诱导的脱颗粒。这被发现与结构性的基础PLD1活性有关，该活性调节磷脂酸的形成，然后控制由免疫受体聚集启动的早期信号。Cbl家族蛋白负向调节酪氨酸激酶偶联受体的信号传递。为了研究c-Cbl和Cbl-b在免疫球蛋白受体信号转导中的作用，从野生型c-Cbl和Cbl-b缺陷小鼠中培养肥大细胞。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转导和炎性介质释放增加。与Cbl-b相反，c-Cbl缺乏对受体诱导的脱颗粒或信号转导没有明显影响。这些结果表明Cbl-b和c-Cbl在免疫受体信号转导中的作用是不同的，Cbl-b而不是c-Cbl是脱颗粒的负调节因子。长期以来，人们一直认为骨髓中存在一种固定的肥大细胞前体，尽管它的鉴定和分离已被证明是困难的。我们用两种肥大细胞特异性抗体进行连续免疫磁分离，从成年小鼠骨髓中纯化和鉴定了一种仅占总细胞数0.02%的系谱决定的肥大细胞前体。我们已经研究了这些细胞的体外生长要求和形态，现在我们正在鉴定在未成熟前体细胞中唯一表达的分子。

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.