Mast cells play an important role in many inflammatory and immunological reactions by releasing an array of mediators. The goal of our studies is to understand the intracellular signal transduction pathways that lead to the release of these molecules. In previous studies, we demonstrated that the protein tyrosine kinase Syk is essential for the high affinity immunoglobulin E receptor (FceRI)-induced degranulation that results in the release of inflammatory mediators. A variant of the rat basophilic RBL-2H3 mast cells that has no detectable Syk was also identified and has been used to examine the structural basis of the regulation of Syk after immune receptor aggregation. These studies identified the linker region of Syk, located between the second SH2 and the kinase domain, as important in regulating the function of this kinase. ? Previously we isolated a mast cell specific monoclonal antibody, mAb BGD6, that recognizes a mast cell precursor present in the bone marrow at very low numbers. By expression cloning using an RBL-2H3 cDNA library and mAb BGD6 a positive clone was identified that showed almost complete identity with FcgRIIB (also called CD32), the low affinity IgG receptor. However, in contrast to the sequence in GenBank, the isolated clone had an insert of 141 bp which codes for an isoform of this receptor previously reported in other species but not in rat cells. This long isoform has an extra 47 amino acids in the cytoplasmic domain. In RBL-2H3 cells both isoforms of FcgRIIB were expressed, with higher expression of the shorter form. Binding studies demonstrated that intact mAb BGD6 bound to both RBL-2H3 and CD32 positive cells, but only the F(ab?)2 bound to the RBL-2H3 cells. Therefore, mAb BGD6 binds to the FcgRIIB only through its Fc portion. On RBL-2H3 cells, the Fab of an anti-CD32 mAb partially inhibited the binding of intact mAb BGD6, but it did not change the binding of the F(ab?)2 fragment of mAb BGD6. These results suggest that the Fc portion of mAb BDG6 contributes to its binding on cells that have FcgRIIB. By this model mAb BGD6 binds through its Fab portion to a cell-surface molecule and the Fc tail interacts with FcgRIIB (CD32).? Immune receptor stimulation results in an increase in intracellular calcium that activates the serine phosphatase calcineurin, which then dephosphorylates the nuclear factor of activated T cells (NFAT). The dephosphorylated NFAT rapidly translocates into the nucleus and induces the transcription of various cytokine genes. NFAT plays an important role in the activation of immune cells such as T, B and mast cells. A plasmid containing the cDNA with three tandem NFAT-binding sites fused to enhanced green fluorescent protein (GFP) was transfected into the RBL-2H3 cells. A cloned cell line was established that became almost totally GFP positive upon FceRI stimulation and had no background of spontaneous GFP expression in non-stimulated cells. These cells are being used in a project to compare the signaling pathways that result in nuclear gene expression from those that induce degranulation and the release of inflammatory mediators. ? Protein tyrosine phosphorylation is one of the earliest detectable events after antigen receptor activation. Recently, we observed that in cultured fetal liver pre-B cells, pre-B cell receptor stimulation induced the strong tyrosine phosphorylation of an 82 kDa protein, whose identity is unclear. The strong phosphorylation of this protein was also observed in antigen-receptor stimulated spleen B cells and mast cells. In mast cells, the phosphorylation of this 82 kDa protein required Syk kinase activity; however, this phosphorylation was independent of the rise of intracellular calcium. Therefore, the tyrosine phosphorylation of this 82 kDa protein directly correlated with early antigen signaling events in mast cells. To identify the 82 kDa phosphorylated protein, lysates from antigen stimulated RBL-2H3 cells were immunoprecipitated with anti-phospho-tyrosine antibody and the 82 kDa phosphorylated band was micro-sequenced. As a control, the phosphorylated 72 kDa band, that is known to contain Syk, was also micro-sequenced. The major component of the 82 kDa band was the hematopoietic cell specific Lyn substrate (HS1), while minor components included cortactin isoform B, inositol polyphosphate 5' phosphatase (SHIP), FYN binding protein, Gab 1, and protein kinase C-delta. The 72 kDa band, contained src homology 2 containing protein of 76 kDa (SLP-76), HS1, Syk, heat shock 70 protein, Btk and SHIP. There were a number of other proteins detected at lower levels in both the 82 and 70 kDa bands. Immunoblotting and immunoprecipitation experiments with control and activated RBL-2H3 cells confirmed that HS1 was the 82 kDa phospho-protein. Therefore, tyrosine phosphorylation of HS1 can serve as a marker for the functional status of early antigen signaling pathways.
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