The degranulation of mast cells in response to ligands that cross-link the high-affinity IgE receptor. FcepsilonR1, is the primary event leading to allergic, asthmatic and anaphylactic reactions. The structural complexity of the FcepsilonR1 (3 subunits), 7 membrane- spanning domains, 4 cytoplasmic tails) has so far defeated attempts to identify essential receptor subunit functional domains, while the large size of the signalling complex in antigen-activated mast cells has so far prevented definition of the minimal assembly of macromolecules required to elicit responses. Dr. Janet Oliver from the University of New Mexico (UNM) and Dr. Eugene Ortega from the Universidad Nacional Autonoma de Mexico (UNAM) conduct complementary research on the molecular mechanisms of signal transduction mediated through the FcepsilonR1 in RBL-2H3 cells, a mast cell model. Dr. Oliver was recently funded (R)! GM-49814-01) to study signal transduction through chimeric receptors that couple the extracellular and trans-membrane domains of an irrelevant receptor to the cytoplasmic tail of a single FcepsilonR1 subunit. These simplified receptors are unique tools to explore the signalling activities of specific FcepsilonR1 subunit cytoplasmic domains. Dr. Ortega is studying signal transduction elicited by three monoclonal antibodies to the FcepsilonR1 alpha/subunit that have similar receptor binding affinities but very different capacities to induce mast cell activation. These antibodies, that cross- link native receptors into dimers but not higher oligomers, represent unique reagents to identify the minimal structure requirements (multiplicity, conformation, lifetimes) and macromolecular composition of a signalling-component receptor complex. The goal of the FIRCA is to use the combined expertise of two laboratories to define the structural, biochemical, ionic and functional properties of monoclonal antibody-induced FcepsilonR1 dimers. Methods developed in Dr. Ortega's laboratory will be used to purify unlabelled and fluorescein-conjugated intact antibodies and antibody fragments, and to analyze the extent of antibody-induced receptor dimerization, the lifetimes of the dimers and the extent of dimer-dimer interactions. Methods developed in Dr. Oliver's laboratory will be used to analyze ionic, biochemical and functional responses to FcepsilonR1 cross-linking (protein-tyrosine phosphorylation, inositol phospholipid turnover, Ca2+ mobilization, secretion, actin assembly, ruffling, adhesion) as well as receptor distribution, endocytosis and interactions with the cytoskeleton. The results of these studies will identify fundamental properties of cross-linked receptors that confer competence to signal. The combined resources of both laboratories is essential to meet the stated aims of the FIRCA proposal. The proposed research complements and enhances, but does not overlap the parent grant R01 GM49814-01.
