Altered patterns of tyrosine phosphorylation and Syk activation for sterically restricted cyclic dimers of IgE-Fc epsilon RI

Previous studies in our laboratory established that the symmetrical bivalent ligand, N,N'-bis[[epsilon-(2,4-dinitrophenyl)amino] caproyl] -L-tyrosyl]-L-cystine ((DCT)(2)-cys), stably cross-links anti-2,4-dinitrophenyl-immunoglobulin E (IgE) bound to high affinity receptors Fc epsilon RI on the surface of RBL-2H3 cells, forming mostly cyclic dimers containing two IgE-Fc epsilon RI and two (DCT)(2)-cys (Posner et al. (1995) J. Immunol. 155, 3601-3509). These cyclic dimers do not trigger Ca2+ or degranulation responses under a variety of conditions. However, we find that the linearly cross-linked IgE-Fc epsilon RI formed at higher concentrations of (DCT)(2)-cys do trigger degranulation in the presence of cytochalasin D, an inhibitor of actin polymerization. We further investigated stimulation by (DCT)(2)-cys of the earliest known events in the functional response, i.e., tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI. At the higher (DCT)(2)-cys concentrations corresponding to linear dimers and maximal degranulation, tyrosine phosphorylation of both beta and gamma are observed. At lower (DCT)(2)-cys concentrations where cross-linking is maximal and cyclic dimers are overwhelmingly dominant, only gamma tyrosine phosphorylation is observed. Cytochalasin D does not affect these phosphorylation patterns, but instead appears to enhance coupling to downstream signaling events. Phosphorylation of Syk occurs at the higher (DCT)(2)-cys concentrations in parallel with beta phosphorylation but does not occur in its absence at the lower (DCT)(2)-cys concentrations. These results suggest that cyclic dimers of IgE-Fc epsilon RI are sterically restricted such that they stimulate tyrosine phosphorylation of gamma but not beta, and this is not sufficient for Syk binding and/or activation.