LIGAND BINDING AND PROTEIN ALTERATIONS IN RBL CELLS

这里提出的实验探索了 免疫系统中的信号转导。 大鼠嗜碱性白血病细胞 系（RBL-2 H3）已被选择用于这些研究。 的交联 这些细胞表面的IgE受体复合物启动了一系列的免疫反应。 导致组胺、5-羟色胺和其他 炎症介质。 从生物化学上讲，这些事件包括钙流， 磷脂甲基化，磷脂酰肌醇周转，瞬时 腺苷酸环化酶激活，可能还有蛋白激酶和蛋白酶 activation. 在生理上，细胞会形成大的表面层， 更紧密地粘附在基质上，增加流体胞饮作用， 嗜碱性颗粒与质膜融合，释放它们的 内容. 本提案将侧重于了解以下方面的早期步骤： RBL-2 H3细胞中的跨膜信号传导，包括 IgE-受体复合物的配体，这些复合物的交联 以及随后细胞骨架组成的变化， 蛋白激酶和其他蛋白修饰酶的活性。 在 特别是：1）将使用荧光分析仪分析配体的结合。 位于洛杉矶的国家流式细胞术资源中心的蛋白质配体 阿拉莫斯国家实验室（NFCR）; 2）细胞蛋白质的变化 包括但不限于配体结合后的细胞骨架 将通过常规方法进行检查，包括PAGE（1和2D），Western 印迹和酶分析（例如肌动蛋白、蛋白激酶）， 化学交联和通过新的原位酶分析;和3） 各种代谢过程的抑制剂将用于确定 这些过程在细胞反应中的重要性。

The experiments proposed here explore certain elements of the mechanism of signal transduction in the immune system. The rat basophilic leukemia cell line (RBL-2H3) has been chosen for these studies. Crosslinking of the IgE-receptor complexes on the surfaces of these cells initiates a series of events leading to the release of histamine, serotonin, and other inflammatory mediators. Biochemically, these events include calcium flux, phospholipid methylation, phosphatidyl inositol turnover, transient adenylate cyclase activation, and probably protein kinase and protease activation. Physiologically, the cells develop large surface lamellae, adhere more tightly to the substrate, increase fluid pinocytosis, and the basophilic granules fuse with the plasma membrane, releasing their contents. This proposal will focus on understanding the early steps of transmembrane signalling in RBL-2H3 cells, including the binding of the ligand to the IgE-receptor complexes, the crosslinking of these complexes and subsequent changes in the composition of the cytoskeleton and in the activity of protein kinases and other protein modifying enzymes. In particular: 1) the binding of ligands will be analyzed using fluorescent protein ligands at the National Flow Cytometry Resource Center at Los Alamos National Laboratory (NFCR); 2) changes in cellular proteins including, but not limited to, the cytoskeleton following ligand binding will be examined by conventional methods including PAGE (1 and 2D), Western blotting, and enzymatic analysis (e.g. for actin, protein kinases), by chemical crosslinking and by novel in situ enzymatic analysis; and 3) inhibitors of various metabolic processes will be used to determine the importance of these processes in the resulting cellular response.