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 in itiating a complex biochemical cascade. We have employed a chemical cr oss-linker in order to stabilize the association of Fc epsilon RI with other cellular proteins that are involved in the early events. We rea cted the water-soluble, membrane-impermeant chemical cross-linker 3,3' -dithiobis(sulfosuccinimidyl propionate) (DTSSP) with permeabilized ra t mucosal mast cells of the RBL line and analyzed immunoprecipitates o f 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 reacte d with DTSSP. Receptors isolated from cells whose receptor-bound IgE h ad been aggregated with antigen prior to cross-linking yielded a simil ar pattern of S-35-labeled proteins. However, when the cells had also been exposed to [gamma-P-32]ATP, the proteins associated with the cros s-linked, aggregated receptors revealed enhanced incorporation of P-32 compared to those associated with cross-linked, unaggregated receptor s. 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) a nd the delta isoform of protein kinase C, were associated with the cro ss-linked Fc epsilon RI in amounts much greater than could be accounte d for by nonspecific cross-linking.