STRUCTURE-FUNCTION RELATIONSHIPS OF THE IGE RECEPTOR

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The work proposed continues the systematic analysis 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. The structural map will be extended by measurements of resonance energy transfer between well defined sites on receptor-bound IgE and cytoplasmically exposed portions of the receptor that are labeled by monoclonal antibodies. Regions of IgE that are critical for its interaction with receptor will be examined by genetic manipulation of the polypeptide chains. Genetic methods will also be used to introduce specific cysteine residues for fluorescent labeling such that energy transfer measurements may be carried out to test the model that IgE undergoes a conformation change upon binding. In addition to providing basic information knowledge of the IgE-receptor interaction may be valuable in clinical treatments of allergies. The interactions between receptors and other cellular components that occur after lgE-receptor complexes have clustered in a triggering configuration will be investigated by fluorescence photobleaching recovery and electrodiffusion measurements of the lateral mobility of fluorescently labeled monoclonal antibodies that dimerize the receptors or are specific for a partially characterized component that appears to associate with receptors. A procedure for measuring rotational mobility by steady-state phosphorescence anisotropy will be developed such that time-dependent changes in the receptor interactions may be monitored under conditions where cell triggering occurs. The particular components that interact with the clustered receptors will be characterized with the use of differential chemical crosslinking, radioactive labeling and purification schemes. The participation of the cytoplasmically exposed parts of the receptor in interactions with other cellular components during signal transduction will be investigated by testing the competence of lgE-receptor complexes with proteolyzed cytoplasmic portions after reconstitution into intact cells and also by microinjection into cells of the monoclonal antibodies specific for cytoplasmic portions. These studies should contribute new insights to understanding the chain-of-events between antigen bridging of lgE-receptor complexes on the cell surface and the degranulation signal.