Altered Patterns of Tyrosine Phosphorylation and Syk Activation for Sterically Restricted Cyclic Dimers of IgE-FcεRI

Abstract

Previous studies in our laboratory established that the symmetrical bivalent ligand, N,N'-bis-[[epsilon-(2,4-dinitrophenyl)amino]caproyl]-L-tyrosyl]-L-cystin e ((DCT)2-cys), stably cross-links anti-2,4-dinitrophenyl-immunoglobulin E (IgE) bound to high affinity receptors Fc epsilonRI on the surface of RBL-2H3 cells, forming mostly cyclic dimers containing two IgE-Fc epsilonRI and two (DCT)2-cys (Posner et al. (1995) J. Immunol. 155, 3601-3609). 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 epsilonRI 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 epsilonRI. 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 epsilonRI 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.

Bivalent Ligands with Rigid Double-Stranded DNA Spacers Reveal Structural Constraints on Signaling by FcεRI

“…Adherent cells were washed and ligands were added in BSS. Unless otherwise indicated, 2 M cytochalasin D was included during stimulation to enhance degranulation stimulated by bivalent ligands (15). After 1 h at 37°C, aliquots of supernatant were taken from each well to assay the extent of ␤-hexosaminidase release from the cells as previously described (15).…”

“…Unless otherwise indicated, 2 M cytochalasin D was included during stimulation to enhance degranulation stimulated by bivalent ligands (15). After 1 h at 37°C, aliquots of supernatant were taken from each well to assay the extent of ␤-hexosaminidase release from the cells as previously described (15). Stimulated release of enzyme activity is expressed as a percentage of the total cellular ␤-hexosaminidase activity present in cell lysates after solubilization in 0.5% Triton X-100.…”

“…Wells were scraped with the pipette tip and lysates were centrifuged for 5 min at 13,000 ϫ g to pellet insoluble debris. Anti-Syk immunoprecipitations were conducted as previously described (15). Aliquots of lysates were mixed with 5ϫ SDS sample buffer (50% glycerol, 0.25 M Tris base (pH 6.8), 5% SDS, 0.5% bromophenol blue) then boiled and centrifuged for 5 min at 13,000 ϫ g. Supernatants were either loaded on a polyacrylamide gel or stored at Ϫ20°C for analysis at a later time.…”

“…In other studies, well-defined bivalent ligands have been used to study the relationship between binding and cross-linking of IgE-Fc⑀RI complexes and subsequent cell activation. In some of these studies, the theoretically predicted extent of equilibrium cross-linking was found satisfactory to account for the biological response (11)(12)(13), whereas, in others, analysis of the data using this theory led to the conclusion that cyclic cross-links formed by some bivalent ligands and bivalent IgE bound to Fc⑀RI can limit effective signaling (14,15). Consistent with these latter findings, Schweitzer-Stenner et al (16) showed that flexible bivalent ligands Ն130 Å in length form cyclic monomers with IgE in solution, whereas bivalent ligands Ͻ45 Å in length efficiently form cyclic dimers.…”

Trivalent Antigens for Degranulation of Mast Cells

“…12 Modeling suggests that cyclic dimers, in which two ligand molecules connect two IgE -FcεRI complexes to form a closed ring, prevent chain elongation, limit the size of ligand-induced aggregates, and may generate an inhibitory signal. 13 In contrast to divalent antigens, trivalent antigens are predicted to form larger, highly branched aggregates ( Figure 1B) leading to strong responses by IgE -FcεRI presenting cells ( Figure 1C). Recently, Whitesides and coworkers 14 reported the preparation of a trivalent antigen capable of aggregating IgE.…”