The binding of a ligand to a cell surface receptor is the first step in a cascade of events that leads to the generation of a transmembrane signal. In many cell systems, simple ligand binding is insufficient to initiate signal transduction, rather, receptor aggregation is required. On the surface of mast cells, basophils and rat basophilic leukemia (RBL) cells are Fc receptors (FceRI) which bind IgE with high affinity. The formation of aggregates of IgE or equivalently of the FceRI receptors triggers various cellular responses. However, it is not clear what physical and steric conditions must be met by the IgE-FceRI aggregates in order to initiate signal transduction. The general goals of this project are to understand receptor aggregation in general and in particular to determine the properties of an IgE-receptor aggregate that are required to initiate particular cellular responses. To do this we use multiparameter flow cytometry to measure multivalent ligand binding and crosslinking of cell surface IgE.
We aim to correlate our crosslinking data with specific cellular pathways such as changes in intracellular Ca2+ levels, tyrosine kinase activity and degranulation.
The aim of this project are to combine experiment and theory to elucidate critical aspects of receptor aggregation. Specifically we will: 1. Employ flow cytometry to experimentally measure the binding parameters (equilibrium and kinetic) of multivalent ligands to cell surface IgE. 2. To develop (and test with our experimental data) mathematical models that predict the time course and equilibrium distribution of IgE aggregation that occur when surface IgE is crosslinked by multivalent ligand. 3. To experimentally measure the time course of cellular pathways (changes in intracellular Ca2+ levels, tyrosine kinase activity and degranulation) initiated by multivalent antigens and to correlate these measurements with our binding and theoretical studies.
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