A process of fundamental importance in immunology is the antigen-mediated crosslinking of cell surface receptors that leads to transmembrane signaling in specialized cells. The critical molecular features of the receptor crosslinking events required to initiate cell activation and the biochemical events that immediately follow are not well understood. The proposed studies advance efforts toward elucidating these events and will build upon a strong foundation of methodologies and concepts from previous studies in this laboratory. These studies will focus on what has proven to be an excellent model system: the high affinity receptor for immunoglobulin E (Fc-epsilon-RI) on RBL-2H3 cells, a mucosal mast cell line. Antigen binding and crosslinking studies will assess a working model that emphasizes the dynamics of crosslinking: maximal responses occur under conditions where antigen-mediated crosslinks are rapidly breaking and reforming such that new receptors are continually being incorporated into the crosslinked aggregate. The model also explains that the reason why most bivalent ligands, which readily crosslink IgE-receptor complexes, do not stimulate cells is because they tend to form highly stable cyclic dimers. The generality of both of these aspects will be examined with a variety of bivalent ligands (including anti-IgE monoclonal antibodies) by comparing detailed binding measurements with assays of biological activity. The possible importance of receptor-receptor orientation will be investigated with measurements of resonance energy transfer. Comparative experiments with a specially developed trivalent ligands and monovalent IgE will allow detailed examination of the relative efficacy of linearly crosslinked receptor aggregates and branched networks of aggregated receptors. Studies of the early events of receptor-mediated cell activation will investigate mechanisms for the initiation and control of the signal transduction cascade. Part of these studies will focus on the central role of tyrosine kinase activation and effects of its inhibition on other pathways. Subcellular preparations will be developed to examine the mechanisms of receptor-mediated tyrosine phosphorylation of cellular and exogenous substrates, and these systems will also be analyzed with steady state kinetics in conjunction with antigen binding measurements. Other studies will examine antigen-mediated desensitization of Fc-epsilon-RI which appears to involve serine and threonine phosphorylation of a receptor subunit and also an unidentified receptor-associated protein. The desensitizing process can be reversed in a time-dependent manner by addition of a monovalent ligand that prevents further antigen-mediated crosslinking of receptors. This process appears to represent a type of immunological memory, and the molecular basis for its mechanism will be examined in conjunction with the antigen binding measurements.
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