Department of Chemistry
Northern Arizona University
The aggregation of cell surface receptors by multivalent ligand underlies many different aspects of cell recognition and response. Most immunologic, hormone and cytokine receptors respond to stimulant with a "bell shaped" dose response curve, an observation that is consistent with aggregation initiating signal transduction. Thus, an important goal in cell biology has been to establish quantitative relationships between the amount of ligand present on a cell surface and the number of crosslinked receptors. A well-studied cellular system that responds to aggregation is the high affinity receptor for IgE (FcRI), which is responsible for initiating allergic reactions. To better understand ligand-induced receptor aggregation in this system, we have been investigating the binding of a model multivalent antigen (DNP-25-PE) to cell-surface anti DNP IgE. To determine the kinetic and equilibrium parameters that characterize crosslinking in this system, we have developed a combined theoretical and experimental approach that is based on multiparameter flow cytometry. Our experimental method allows for the simultaneous measurement of both the number of ligand molecules bound per cell and the number of receptor binding sites that are occupied per cell. The results indicate how IgE-FcRI aggregation depends on the total concentrations of DNP-25-PE and surface IgE. As expected, we find that maximal aggregation occurs at an optimal antigen concentration (the crosslinking curves are "bell shaped") and that receptor aggregation varies with the total concentration of surface IgE as predicted by theoretical studies. We then measure the dynamics of crosslinking in real time and correlate aggregation with cell signaling.