Swaim W D, Minoguchi K, Oliver C, Hamawy M M, Kihara H, Stephan V, Berenstein E H, Siraganian R P
Laboratory of Immunology, NIDR, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Jul 29;269(30):19466-73.
The monoclonal antibody AA4 (mAb AA4) recognizes novel alpha-galactosyl derivatives of GD1b on rat basophilic leukemia (RBL-2H3) cells. The binding of mAb AA4 induced protein tyrosine phosphorylations without histamine release. Several of the same proteins including Fc epsilon RI beta, Fc epsilon RI gamma, p72syk, and phospholipase C-gamma 1 were tyrosine-phosphorylated by mAb AA4 binding and by the activation of the high affinity IgE receptor, Fc epsilon RI. There was also activation of the p53/56lyn and p72syk protein-tyrosine kinases, but compared to direct Fc epsilon RI activation, mAb AA4 did not result in increased tyrosine phosphorylation of pp105-115 or pp125FAK, and the receptor subunits (Fc epsilon RI beta and Fc epsilon RI gamma) were more heavily phosphorylated. Furthermore, the time course of the phosphorylations with mAb AA4 was slower than that induced by Fc epsilon RI aggregation. By immunofluorescence, the tyrosine-phosphorylated proteins after mAb AA4 stimulation were localized in patches at the cell membrane and in areas of cell-cell contact, whereas after Fc epsilon RI activation, there was a reticular cytoplasmic pattern. There were no protein tyrosine phosphorylations either when Fc epsilon RI was saturated with IgE or when F(ab')2 fragments of mAb AA4 were used, although the F(ab')2 fragments still induced morphological changes. There was also coprecipitation of the beta and gamma subunits of Fc epsilon RI with the anti-ganglioside antibody. These data strongly suggest the involvement of Fc epsilon RI in the antiganglioside-induced protein tyrosine phosphorylations. Moreover, phosphorylations of these proteins including the beta and gamma chains of Fc epsilon RI and activation of p53/56lyn and p72syk did not result in histamine release.
单克隆抗体AA4（mAb AA4）可识别大鼠嗜碱性白血病（RBL - 2H3）细胞上GD1b的新型α - 半乳糖基衍生物。mAb AA4的结合可诱导蛋白酪氨酸磷酸化，但不引起组胺释放。包括FcεRIβ、FcεRIγ、p72syk和磷脂酶C - γ1在内的几种相同蛋白，可通过mAb AA4结合以及高亲和力IgE受体FcεRI的激活而发生酪氨酸磷酸化。p53/56lyn和p72syk蛋白酪氨酸激酶也被激活，但与直接激活FcεRI相比，mAb AA4并未导致pp105 - 115或pp125FAK的酪氨酸磷酸化增加，且受体亚基（FcεRIβ和FcεRIγ）的磷酸化程度更高。此外，mAb AA4诱导磷酸化的时间进程比FcεRI聚集诱导的要慢。通过免疫荧光观察，mAb AA4刺激后酪氨酸磷酸化的蛋白定位于细胞膜上的斑块以及细胞 - 细胞接触区域，而FcεRI激活后则呈现网状细胞质模式。当FcεRI被IgE饱和或使用mAb AA4的F(ab')2片段时，均未发生蛋白酪氨酸磷酸化，尽管F(ab')2片段仍可诱导形态学变化。FcεRI的β和γ亚基也与抗神经节苷脂抗体发生共沉淀。这些数据强烈表明FcεRI参与了抗神经节苷脂诱导的蛋白酪氨酸磷酸化。此外，包括FcεRI的β和γ链在内的这些蛋白的磷酸化以及p53/56lyn和p72syk的激活并未导致组胺释放。
