Altered patterns of tyrosine phosphorylation and Syk activation for sterically restricted cyclic dimers of IgE-Fc epsilonRI.
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.
Harris
N T
NT
Department of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Goldstein
B
B
Holowka
D
D
Baird
B
B
D002460:Cell Line; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D007139:Immunoglobulin epsilon-Chains / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017455:Receptors, IgE / Q000378:metabolism*; D015398:Signal Transduction; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism*
D004792:Enzyme Precursors; D007139:Immunoglobulin epsilon-Chains; D047908:Intracellular Signaling Peptides and Proteins; D017455:Receptors, IgE; D007073:Immunoglobulin E; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase