The proposed studies will advance elucidation of the features of antigen-mediated crosslinking of lgE bound to its high affinity receptor, Fc epsilonRI, on mast cells that are critical for initiating a cascade of signaling events leading to the release of key modiators in allergic responses. The studies will build on strong foundation of fluorescence and other methodologies and concepts that are derived from previous work on this well-characterized model immune cell system. We have established that a small, symmetrical bivalent ligand stimulates a cellular response depending on whether this ligand forms small cyclic complexes or linear chains with lgE bound to Fc epsilon RI , providing evidence that structural restrictions between crosslinked lgE-Fc epsilon RI can limit cell activation. This hypothesis will be tested in Specific Aim 1 with a new generation of rigid bivalent ligands made with double-stranded DNA, together with a specially prepared bi- specific lgE. The structural arrangement of crosslinked lgE-Fc epsilon Rl beta and gamma subunits by Lyn, and the binding and activation of the tyrosine kinase Syk. Binding of trivalent and multivalent antigens will also be analyzed in conjunction with their functional activities.
Specific Aim 2 will investigate the functional importance of specialized plasma membrane domains that are being characterized in this laboratory. Crosslinking of lgE-Fc epsilon Ri causes association with detergent-resistant membrane domains that contain abundant Lyn tyrosine kinase activity, and our recent results indicate that these interactions are important for initiating Fc epsilon Rl mediated signal transduction. These domain-Fc epsilon Rl interactions, their regulation by the mirofilament cytoskeleton, and the consequences of these on early and late signaling events will be investigated in biochemical preparations and intact cells. Because of the central role of ligand binding and receptor aggregation in immune cell activation and the accumulating evidence that signaling molecules localized to membrane domains are integrally involved, the proposed investigation should lead to new concepts and targets for therapeutic intervention.
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