Purified rat serosal mast cells were sensitized with mouse immunoglobulin E (IgE) anti-2,4-dinitrophenyl antibody, partially depleted of phosphate, labeled with [32P]orthophosphate, and stimulated with dinitrophenylated bovine serum albumin or control protein. After 15-120 seconds at 37C, the cells were extracted with nonionic detergent. IgE receptors were purified by repetitive affinity chromatography and were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and radioautography. Antigenic stimulation of intact rat mast cells produced a rapid and marked increase in the phosphorylation of the surface-exposed a component of the IgE receptor. However, phosphorylation of the 33,000 Mr (3 component of the IgE receptor was not altered significantly by antigen stimulation. This suggests that the selective increase in phosphorylation of the IgE receptor a component may be part of the physiologic mediator secretion process triggered by antigen.Mast cells and basophils have cell-surface receptors that bind IgE with high affinity. Each IgE receptor is composed of a surface-exposed glycoprotein (1, 2), referred to as the a component in recent terminology (3), and a nonglycosylated protein (4, 5) termed the ,B component. Tryptic peptide mapping studies indicate that each component is a distinct polypeptide (6). The a component of rat mast cells, Mr 45,000 (7), is somewhat smaller than the a component of rat leukemic basophils analyzed in the same NaDodSO4/polyacrylamide gel system (6), whereas P components of both cell types are indistinguishable in size (Mr 33,000) (6, 7).The apposition of adjacent receptors on rat mast cells is the necessary and sufficient signal (8) for cell activation leading to the noncytotoxic release and generation of a variety of cellular mediators. Mast cell activation has been associated with changes in diacylglycerol levels (9), phospholipid methylation (10), cyclic AMP levels (11), influx of calcium (10), and protein phosphorylation (12). We have demonstrated that the IgE receptor a component of rat mast cells is a phosphoprotein and that its phosphorylation is significantly increased by a nonimmunologic stimulus of histamine release (7). In this study, we demonstrate that an immunologic stimulus with antigen produces a rapid and selective increase in phosphorylation of the a component of the rat mast cell IgE receptor. Thus, it seems possible that increased phosphorylation of the receptor a component is part of the antigen-stimulated process leading to mediator secretion.
MATERIALS AND METHODSAntigen and IgE Antibody. The monoclonal 2,4-dinitrophenyl (DNP)-specific murine IgE antibody H1 DNP-E-26 (13) was isolated from ascites from the hybridoma made available by Fu-Tong Liu and David Katz. Protein soluble in 30% saturated ammonium sulfate was precipitated by 60% saturated ammonium sulfate (13) and was further purified by gel filtration on Sepharose CL-6B. Based on polyacrylamide gel electrophoresis in NaDodSO4, the IgE was >95% pure. DNP-treated bovine serum albumin (DNP-albumin...