Function Analysis of receotor aggregation in cell signaling

用共聚焦荧光显微镜观察抗原刺激后大鼠嗜碱粒细胞系(RBL-2H3)表面CD63抗原的表达。CD63抗原定位于嗜碱性粒细胞、肥大细胞和血小板的嗜碱性颗粒膜上。CD63抗原的表面表达反映了RBL-2H3细胞的脱颗粒。我在P815肥大细胞瘤细胞中做了同样的实验，这些细胞都带有转基因的IgE受体。在具有正常IgE受体的P815细胞中可观察到该基因的表达，而在缺失β或γ亚基C末端胞质结构域的P815变异细胞中未观察到该表达。此外，在具有正常IgE受体的P815细胞中，大部分表达被除草剂A阻断。结果表明，β和γ亚基C末端胞质结构域的酪氨酸磷酸化是脱颗粒所必需的。接下来，我制备了针对SH2结构域高度保守序列(GTFLVRESETTK)的单抗。针对多肽标记的锁孔帽状血蓝蛋白制备的小鼠IgG1(12E和32D)与抗原肽特异性结合，但不与载体蛋白结合。Western印迹分析表明，一种IgG1(12E)主要识别RBL-2H3细胞Triton X-100提取物中的62 kDa蛋白质(可能是src家族酪氨酸激酶)，另一种(32D)主要识别32 kDa和110 kDa蛋白质。共聚焦荧光显微镜显示SH2结构域呈弥漫性胞浆分布，在细胞核内不存在。经抗原刺激后，12E和32D免疫球蛋白染色的细胞分布明显不同，12E免疫球蛋白在细胞膜上呈强阳性，而32D免疫球蛋白在胞浆内呈小颗粒染色。当12E免疫球蛋白与62 kDa蛋白结合时，结果表明酪氨酸激酶沿着质膜聚集和/或在抗原刺激后结构域发生构象变化。

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.