LIGAND BINDING AND PROTEIN ALTERATIONS IN RBL CELLS

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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.