Characterization Of Cell Surface Molecules Important For

高亲和力免疫球蛋白E （IgE）受体（FcepsilonRI）聚集，在肥大细胞和嗜碱性细胞上表达，立即引发超敏反应。据报道，聚集的FcepsilonRI在RBL-2H3细胞中迅速迁移到脂筏中。我们证实在细胞裂解物的脂筏部分发现了聚集的FcepsilonRI。此外，我们表明，交联的FcepsilonRI在内化后仍然与耐洗涤剂结构相关。先前的形态学研究报道，聚集的FcepsilonRI通过网格蛋白包被的凹坑被内吞，而凹坑通常与脂质筏无关。为了解决这种明显的差异，我们使用siRNA来抑制网格蛋白介导的内化机制成分的表达，即网格蛋白重链和AP-2 （α -适应蛋白或mu2亚基）。转铁蛋白受体（TfR）通过网格蛋白介导的过程内吞，正如预期的那样，每个转染的siRNA导致TfR表面表达升高2至3倍，几乎完全抑制其内吞作用。相反，对FcRI的表面表达水平和二硝基苯-人血清白蛋白(DNP-HSA)/IgE/FcepsilonRI复合物的内吞作用没有影响。相反，DNP-HSA/IgE/FcepsilonRI的内化被过表达一个显性阴性的dynamin突变体所抑制。我们得出结论，交联FcepsilonRI的内化不需要AP-2/网格蛋白复合物，而是动力蛋白依赖的，可能是脂质筏介导的。

Aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcepsilonRI), expressed on mast cells and basophils, initiates the immediate hypersensitivity reaction. Aggregated FcepsilonRI has been reported to rapidly migrate to lipid rafts in RBL-2H3 cells. We confirmed that aggregated FcepsilonRI is found in the lipid raft fractions of cellular lysates. Furthermore, we show that the cross-linked FcepsilonRI remains associated with detergent-resistant structures upon internalization. Previous morphological studies have reported that aggregated FcepsilonRI is endocytosed via clathrin-coated pits, which in general are not lipid raft associated. To address this apparent discrepancy, we employed siRNA to suppress expression of components of the clathrin-mediated internalization machinery, namely, clathrin heavy chain, and the AP-2 (alpha-adaptin or mu2-subunit). Transferrin receptor (TfR) is endocytosed by a clathrin-mediated process and, as expected, each transfected siRNA caused a two to threefold elevation of TfR surface expression and almost completely inhibited its endocytosis. In contrast, there was no effect on surface expression levels of FcRI nor on the endocytosis of the dinitrophenyl-human serum albumin (DNP-HSA)/IgE/FcepsilonRI complex. On the contrary, internalization of DNP-HSA/IgE/FcepsilonRI was inhibited by overexpression of a dominant-negative dynamin mutant. We conclude that internalization of cross-linked FcepsilonRI does not require the AP-2/clathrin complex but is dynamin-dependent and may be lipid raft mediated.