Structure-Function Relationships of Immunoreceptors

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DESCRIPTION (provided by applicant): This research is aimed at a fundamental understanding of key processes in immune cell activation via multichain antigen receptors, and it is focused on the receptor for IgE on mast cells, FceRI, as a well characterized prototype that plays a central role in the allergic immune response. Proposed studies will investigate plasma membrane heterogeneity in FceRI signaling, and the participation of liquid ordered regions of the plasma membrane commonly called "lipid rafts." Specific Aim 1 is to characterize dynamic interactions between FceRI and tyrosine kinases Lyn and Syk as well as other signaling proteins that occur after IgE-FceRI are crosslinked by multivalent ligands to activate the cells. Molecular interactions are observed with dual color fluorescence correlation spectroscopy, and the structural bases for these will be probed. Specific Aim 2 is to determine how the cell targets responses in spatial relationship to clustered FceRI and to decipher the biophysical and membrane trafficking bases for pronounced membrane heterogeneity. Recently developed methods for micropatterning of ligands reveal striking differences in the reorganization of inner and outer leaflet components of the plasma membrane. Patterning on finer scales should enable imaging at hundred-nanometer resolution the molecular redistributions that are spatially targeted in the membrane. Specific Aim 3 is to investigate the role of the plasma membrane-associated cytoskeleton in regulating the size and dynamics of liquid ordered/liquid disordered lipid phase separations that are hypothesized to underlie the functional consequences of rafts. Complementary methods of fluorescence resonance energy transfer, electron spin resonance, and mass spectrometry will be used to evaluate effects of agents that perturb plasma membrane homeostasis and function. These investigations integrate physical, chemical and biological approaches to provide new insights into plasma membrane structure and dynamics and their roles in immune cell signaling.