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 [35S]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 35S-labeled proteins. However, when the cells had also been exposed to [gamma-32P]ATP, the proteins associated with the cross-linked, aggregated receptors revealed enhanced incorporation of 32P 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 p53/56lyn 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.
Mao
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SY
Section on Chemical Immunology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892-1820.
Yamashita
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T
Metzger
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H
D000818:Animals; D015153:Blotting, Western; D002463:Cell Membrane Permeability; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D007073:Immunoglobulin E / Q000737:chemistry / Q000378:metabolism*; D007163:Immunosorbent Techniques; D015471:Leukemia, Basophilic, Acute; D046911:Macromolecular Substances; D008407:Mast Cells / Q000276:immunology*; D010766:Phosphorylation; D019000:Phosphotyrosine; D051381:Rats; D017455:Receptors, IgE / Q000737:chemistry* / Q000378:metabolism*; D013388:Succinimides; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D017455:Receptors, IgE; D013388:Succinimides; D019000:Phosphotyrosine; D007073:Immunoglobulin E; D014443:Tyrosine; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate)