CHEMICAL CROSS-LINKING OF IGE-RECEPTOR COMPLEXES IN RBL-2H3 CELLS

Aggregation of the high-affinity receptors for IgE (FC epsilon RI) on mast cells activates nonreceptor kinases and other enzymes, thereby initiating a complex biochemical cascade. We have employed a chemical cross-linker in order to stabilize the association of Fc epsilon RI with other cellular proteins that are involved in the early events. We reacted the water-soluble, membrane-impermeant chemical cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) with permeabilized rat mucosal mast cells of the RBL line and analyzed immunoprecipitates of the receptor in detergent lysates of cells that had biosynthetically incorporated [S-35]cysteine. Gel electrophoresis revealed substantial amounts of nonreceptor components only when the cells had been reacted with DTSSP. Receptors isolated from cells whose receptor-bound IgE had been aggregated with antigen prior to cross-linking yielded a similar pattern of S-35-labeled proteins. However, when the cells had also been exposed to [gamma-P-32]ATP, the proteins associated with the cross-linked, aggregated receptors revealed enhanced incorporation of P-32 compared to those associated with cross-linked, unaggregated receptors. A variety of antibodies were used to try to identify the associated proteins. Of those tested for, two, the src-like kinase p53/56(lyn) and the delta isoform of protein kinase C, were associated with the cross-linked Fc epsilon RI in amounts much greater than could be accounted for by nonspecific cross-linking.