Characterization Of Cell Surface Molecules Important For

肥大细胞和嗜碱性粒细胞上表达的高亲和力免疫球蛋白E（IgE）受体（Fc ε RI）的聚集引发速发型超敏反应。已报道聚集的Fc ε RI在RBL-2 H3细胞中快速迁移至脂筏。我们证实，聚集的Fc ε RI被发现在细胞裂解物的脂筏部分。此外，我们表明，交联的FcepsilonRI仍然与耐洗涤剂的结构后内化。先前的形态学研究已经报道，聚集的FcepsilonRI通过网格蛋白包被的小凹被内吞，其通常与脂筏无关。为了解决这一明显的差异，我们采用siRNA来抑制网格蛋白介导的内化机制的组分的表达，即网格蛋白重链和AP-2（α-适配蛋白或μ 2亚基）。转铁蛋白受体（TfR）通过网格蛋白介导的过程被内吞，并且如预期的，每个转染的siRNA引起TfR表面表达的两到三倍升高，并且几乎完全抑制其内吞。相反，对FcRI的表面表达水平没有影响，对二硝基苯-人血清白蛋白（DNP-HSA）/IgE/FcepsilonRI复合物的内吞作用也没有影响。相反，DNP-HSA/IgE/FcepsilonRI的内化被显性负性发动蛋白突变体的过表达抑制。我们的结论是，内化交联FcepsilonRI不需要AP-2/网格蛋白复合物，但动力蛋白依赖，可能是脂筏介导的。

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.