LIGAND BINDING AND PROTEIN ALTERATIONS IN RBL CELLS

这里提出的实验探索了某些元素的机制 免疫系统中的信号转导。大鼠嗜碱性白血病细胞 我们选择了品系(RBL-2H3)进行这些研究。交联剂的合成 这些细胞表面的免疫球蛋白受体复合体启动了一系列 导致组胺、5-羟色胺和其他物质释放的事件 炎症介质。在生物化学方面，这些事件包括钙流动， 磷脂甲基化，磷脂酰肌醇转化，一过性 腺苷环化酶激活，可能还有蛋白激酶和蛋白酶 激活。从生理上讲，细胞发育出大的表面层， 更紧密地附着在底物上，增加液体吞噬作用，并且 嗜碱性颗粒与质膜融合，释放它们的 内容。这项提案将侧重于了解 RBL-2H3细胞中的跨膜信号转导，包括结合 Ig E受体复合体的配体，这些复合体的交联性 以及随后细胞骨架的组成和细胞内 蛋白激酶和其他蛋白质修饰酶的活性。在……里面 具体内容：1)用荧光法分析配体的结合 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，包括第(1和2D)页，Western 印迹和酶分析(例如肌动蛋白、蛋白激酶)，通过 化学交联和新的原位酶分析；3) 各种代谢过程的抑制剂将被用来确定 这些过程在由此产生的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.