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RI in Relationship to the Stimulated Tyrosine Phosphorylation of FcRI1
*
Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY 14853 and
Theoretical Biology and Biophysics Group, Theoretical Division, Los Alamos National Laboratory, Los Alamos, NM 87545.
Multivalent DNP-BSA is commonly used to cross-link anti-DNP IgE
bound to Fc
RI to stimulate cellular responses, although key features
of the binding process are unknown. Fluorescence quenching can be used
to study the kinetics of DNP-BSA binding to FITC-IgE. We observe that
DNP-BSA binds more slowly to IgE than does an equimolar amount of a
monovalent DNP ligand, suggesting that the average effective number of
DNP groups per BSA is less than one. The binding data are well
described by a transient hapten exposure model in which most of the DNP
groups are unavailable for binding but have some probability of
becoming exposed and available for binding during the time of the
binding measurement. Additional experiments indicate that, for
suboptimal to optimal concentrations of DNP-BSA, most of the FITC
fluorescence quenching on the cell surface is due to cross-linking
events. With these concentrations at 15°C, the kinetics of FITC
fluorescence quenching by DNP-BSA correlates with the kinetics of
DNP-BSA-stimulated tyrosine phosphorylation of FcRI. At 35°C, the
phosphorylation kinetics are biphasic during the time period in which
cross-linking continues to increase. Our results establish a
quantitative relationship between the timecourse for cross-linking by
multivalent Ag and FcRI-mediated signaling, and they provide the
means to predict the kinetics of cross-linking under a wide variety of
conditions.
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