Functional contributions of the FcepsilonRIalpha and FepsilonRIgamma subunit domains in FcepsilonRI-mediated signaling in mast cells.

Abstract

The functional contributions of the alpha and gamma subunit domains of the high affinity receptor for IgE (Fcepsilon-RI) were determined following chimeric receptor aggregation. Chimeric receptors of the extracellular (EC) and cytoplasmic tail (CT) domains of FcepsilonRI and the IL-2R p55 subunit (I) were constructed and stably expressed in RBL-2H3 cells. Signaling (inositol phosphate production, tyrosine phosphorylation, Ca2+ mobilization, and secretion of histamine and arachidonic acid metabolites) via alpha/gamma/gamma or I/gamma/gamma was similar to the native rat receptor, and both were shown to associate with endogenous FcepsilonRIbeta and FcepsilonRIgamma subunits. Therefore, the contributions of the EC domains could not be evaluated. The chimeras alpha/I/gamma and I/I/gamma were found to be single polypeptide chains, as they did not associate with beta and gamma. Signaling via alpha/I/gamma resulted in the appearance of biochemical events common to the native receptor. Cross-linking I/I/gamma elicited histamine release, [14C]arachidonic acid metabolites, tyrosine phosphorylation, Ca2+ mobilization, and only inositol trisphosphate production, which were not of a similar magnitude to the native FcepsilonRI. No biochemical events were elicited by cross-linking alpha/I/I or I/I/I. These results demonstrate that both the FcepsilonRIalpha EC domain and the FcepsilonRIgamma CT domain are essential for the FcepsilonRI signaling process, and that while FcepsilonRIIgamma CT plays a critical role in FepsilonRI signaling, the EC domain of FcepsilonRIalpha has a major contribution in signaling, as well as a role in modulating the magnitude of the biochemical events.

Generation, Isolation, and Maintenance of Rodent Mast Cells and Mast Cell Lines

“…The RBL‐2H3 cell line proved to be invaluable for the delineation of the structure and immediate signaling events associated with the FcɛRI (Kinet et al, ; Beaven and Metzger, ). In addition, RBL‐2H3 cells could be readily transfected, which allowed the generation of cell lines expressing the human FcɛRI and other native and chimeric receptors (Gilfillan et al, ; Repetto et al, ). This not only allowed for the delineation of the role for specific molecules and the contribution of specific receptor domains to the initiation of signaling, but also provided the pharmaceutical industry with a suitable model for high‐throughput screening of potential inhibitors of mast cell activation.…”