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。我们研究的目的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对于免疫受体诱导的脱粒是必不可少的，脱粒导致炎症介质的释放。还鉴定了大鼠嗜碱性RBL-2H3肥大细胞的变体，其没有可检测到的Syk，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Syk的连接区，位于第二个SH2和激酶结构域之间，在调节这种激酶的功能中很重要。为了鉴定在信号传导中至关重要的蛋白酪氨酸激酶林恩和Syk的新底物，我们筛选了从RBL-2H3细胞制备的cDNA表达文库中的体外酪氨酸磷酸化蛋白。鉴定了5个克隆作为潜在的林恩底物和8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，在RBL-2H3细胞中稳定过表达野生型或无催化活性形式的PLD 1或PLD 2。这种受体诱导的PLD激活需要蛋白酪氨酸激酶Syk，并导致PLD 1和PLD 2的激活。然而，PLD 1是大多数受体诱导的PLD活性的来源。有增强的受体诱导的脱粒仅在过表达催化失活的PLD 1的细胞。这被发现与组成型基础PLD 1活性相关，该活性调节磷脂酸形成，然后控制由免疫受体聚集引发的早期信号。Cbl家族蛋白负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缺陷型小鼠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具有不同的作用，并且Cbl-b而不是c-Cbl作为脱粒的负调节剂起作用。长期以来，人们一直认为骨髓中存在一种定向肥大细胞前体，尽管其鉴定和分离已被证明是困难的。我们已经使用了连续免疫磁性分离与两个肥大细胞特异性抗体纯化和表征谱系承诺肥大细胞前体从成年小鼠骨髓中，仅占总细胞的0.02%。我们已经研究了这些细胞的体外生长要求和形态，我们现在正在表征在未成熟前体中独特表达的分子。

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.