The structure and cell surface dynamics of two different immunoreceptors, the high affinity receptor for IgE (Fc epsilon-Rl) and the T cell receptor for antigen (TCR), will be investigated in a continuing effort to elucidate the molecular details of how these complex proteins function to initiate a cascade of intracellular events in response to foreign antigen. Fluorescence resonance energy transfer will be used to map the spatial relationships among subunits within TCR and between this receptor and other proteins with which it interacts. These studies will include the development of novel energy transfer probes based on genetically engineered, covalently coupled heavy and light chain variable domains from monoclonal anti-receptor antibodies to obtain more precise distance information. Previous energy transfer measurements showed the conformation of IgE to be bent, and this structure will be further characterized with electron microscopy. Chimeric murine IgE/ human IgG1 antibodies will be used to identify the sequences involved in maintaining the bent conformation, and computational methods will be employed to assess the conformation of the segment of seven amino acids at the proposed site of bending and interaction with Fc epsilon-RI. Biochemical methods will be used to characterize proteins that interact with the IgE-receptor complex and are captured by chemical crosslinking. Efficient cell-cell fusion will be exploited as a method for investigating receptor function with both mutant and native receptors. Phosphorescence anisotropy decay measurements of monomeric and dimeric IgE-receptor complexes will be continued to investigate the structural interactions resulting in the reduced rotational diffusion observed for dimers; wildtype Fc epsilon-RI will be compared to mutants with cytoplasmic tail deletions and to chimeric constructs of the alpha subunit, and anti-Fc epsilon-RI monoclonal antibodies will be used to directly assess the influence of bound IgE on the receptor mobility properties. The dynamics of TCR on the cell surface will also be characterized with measurements of fluorescence photobleaching recovery, phosphorescence anisotropy decay, and quantitative video microscopy. Mutant forms of the TCR will be examined to see whether TCR dynamics play an important role in its functional interactions with histocompatibility antigens on target cells.
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