STRUCTURE - FUNCTION RELATIONSHIPS OF THE IGE RECEPTOR

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The work proposed is a continuation of analyses of the three-dimensional structure of the immunoglobulin E(IgE)-receptor complex, its interactions with other cellular components, and the mechanism by which it transmits a signal across the plasma membrane of mast cells and basophils to trigger degranulation in allergic responses. Structural mapping of distances between well-defined sites in IgE, the receptor, and the membrane surface will be continued using resonance energy transfer methods, and a new approach to this technique will be developed using fluorescently-labeld monoclonal antibodies specific for sites in the IgE-receptor complex. Functional effects of the monoclonals on cellular degranulation will be tested and correlated to their structural locations. Biochemical studies on the membrane -bound receptor will be aimed at extending previous information about the cytoplasmic exposure of the Beta and Gamma subunits by examining the susceptibility of membrane-bound receptor to limited proteolytic digestion. Other biochemical studies on the structure of the IgE-receptor complex will include an attempt to photoaffinity label a potential protein kinase site on the receptor and efforts to isolate and Fc fragment of rodent IgE that binds to the receptor. An understanding of the details of the interaction of IgE with the receptor should ultimately aid in the development of receptor-specific anti-allergic pharmaceuticals. Recent observations of large-scale clustering of receptors on the cell surface that is induced by small oligomers of IgE will be extended by making quantitative measurements with fluorescence photobleaching recovery and resonance energy transfer methods. These approaches should permit analysis of the kinetics of the clustering under conditions that are relevant to degranulation such that the temporal relationship between two processes may be assessed. Biochemical studies will be directed toward the identification of cellular components that interact with clustered receptors and thereby may be involved in the transmembrane signal. For these purposes chemical cross-linking and selective cytoskeletal depolymerigation methods will be used to isolate receptor-associated components from detergent-solubilized cells. These studies should contribute new insights to structural and functional chain-of-events between antigen bridging of IgE-receptor complexes on the cell surface and the degranulation signal.