Function Analysis of receotor aggregation in cell signaling

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The surface expression of CD63 antigen in rat basophilic leukemia cells (RBL-2H3) was observed after antigen stimulation by confocal fluorescence microscopy. CD63 antigen located on the basophilic granule membranes in resting basophils, mast cells and platelets. The surface expression of CD63 antigen reflected the degranulation in RBL-2H3 cells. I did the same experiments in P815 mastocytoma cells with transfected IgE receptors. The expression was observed in P815 cells with normal IgE receptors, but not in P815 variant cells with IgE receptors which missing a C-terminal cytoplasmic domain of beta or gamma subunit. In addition, the expression in P815 cells with normal IgE receptors was mostly blocked by the pretreatment of herbimycin A.The results suggested that tyrosine phosphorylation of the C-terminal cytoplasmic domains of beta and gamma subunits was essential for degranulation.Next, I have prepared monoclonal antibodies for a highly conserved sequence (GTFLVRESETTK) in SH2 domains. Mouse IgGls (12E and 32D) prepared against a peptide-conjugated keyhole limpet hemocyanin specifically bound the antigenic peptide but not the carrier protein. Western blot analysis showed that one IgGl (12E) recognized mainly 62kDa proteins (possibly src-family tyrosine kinase) from triton X-100 extracts of RBL-2H3 cells and that another (32D) recognized mainly 32 and 110 kDa proteins. Confocal fluorescence microscopy showed that the SH2 domains had a diffuse cytoplasmic distribution and were not present in the nucleus. Following antigen stimulation, a markedly different cellular distribution was observed in the cells stained with 12E and 32D IgGs.12E IgGs strongly stained the plasma membranes while 32D IgGs stained small granules in the cytoplasm. As 12E IgGs bound 62 kDa proteins on Western blotting, the results suggested that tyrosine kinases cluster along the plasma membranes and/or that conformational changes occur in the domains after antigen stimulation.