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Function Analysis of receotor aggregation in cell signaling

受体聚集在细胞信号传导中的功能分析

基本信息

批准号：
07672373
负责人：
FURUNO Tadahide
金额：
$ 1.28万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672373/
关键词：
signal transduction receptor calcium ion concentration imaging analysis B cell basophil hapten antigen

项目摘要

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.

用共聚焦荧光显微镜观察了抗原刺激后大鼠嗜碱性白血病细胞（RBL-2 H3）表面CD 63抗原的表达。CD 63抗原定位于静息嗜碱性粒细胞、肥大细胞和血小板的嗜碱性颗粒膜上。RBL-2 H3细胞表面CD 63抗原的表达反映了细胞的脱颗粒。我用转染IgE受体的P815肥大细胞瘤细胞做了同样的实验。在IgE受体正常的P815细胞中观察到该表达，而在IgE受体缺失β或γ亚基C端胞质结构域的P815变异细胞中未观察到该表达。此外，在P815细胞与正常的IgE受体的表达主要是由除草霉素A的预处理阻断。结果表明，酪氨酸磷酸化的β和γ亚基的C-末端胞质结构域是必不可少的脱粒。接下来，我已经准备了一个高度保守的序列（GTFLVRESETTK）在SH 2域的单克隆抗体。针对肽缀合的钥孔血蓝蛋白制备的小鼠IgG（12 E和32 D）特异性结合抗原肽，但不结合载体蛋白。Western印迹分析显示，一个IgGl（12 E）主要识别来自RBL-2 H3细胞的triton X-100提取物的62 kDa蛋白（可能是src家族酪氨酸激酶），而另一个（32 D）主要识别32和110 kDa蛋白。共聚焦荧光显微镜显示，SH 2结构域有一个弥漫性的细胞质分布，并不存在于细胞核中。抗原刺激后，一个显着不同的细胞分布中观察到的细胞与12 E和32 D IgG。12 E IgG强烈染色的质膜，而32 D IgG染色的细胞质中的小颗粒。由于12 E IgG在Western印迹上结合62 kDa蛋白，结果表明酪氨酸激酶沿质膜沿着聚集和/或抗原刺激后结构域发生构象变化。