Ran S, Loyter A, Rivnay B
Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
Biochemistry. 1989 Jan 24;28(2):644-51. doi: 10.1021/bi00428a034.
The high-affinity receptor for IgE (Fc epsilon R) is the cellular trigger of the antigen-induced activation of mast cells and basophils. To examine the functional integrity of Fc epsilon R, we have adopted a protein implantation procedure whereby the purified receptor complex was coreconstituted with Sendai virus envelopes. The latter promoted fusion of the hybrid vesicles with recipient cells such as rat basophilic leukemia, RBL-2H3, thus serving as a vehicle for the receptor. The implanted Fc epsilon R was complexed with 125I-labeled mouse IgE (anti-DNP) to permit receptor quantification as well as specific triggering by DNP20BSA. Implantation in the presence of unlabeled rat IgE, which blocked the native receptors on the recipient RBL-2H3 cells, resulted in incorporation of up to 15 ng of receptor-bound IgE/10(6) cells. This was roughly equivalent in amount to 10-20% of the native receptors on such cells. The exocytosis which was triggered in the recipient cells by reagents that specifically recognized the implanted IgE reached between 15 and 50% of the maximal response. Various treatments that interfered with the activities of the viral envelopes reduced both receptor incorporation (3-5-fold) and cell degranulation (3-10-fold). These included separation of the receptor from the reconstituted envelopes, addition of serum to the incubation mixture (to inhibit vesicle-cell binding), and trypsinization of the virus (to inhibit vesicle-cell fusion). Poly(ethylene glycol) 8000 (4%) enhanced both the incorporation of the receptor and its functional responses. These treatments distinguished between real incorporation of IgE-Fc epsilon R complexes and other mechanisms of 125I-IgE association with the recipient cells.(ABSTRACT TRUNCATED AT 250 WORDS)
IgE高亲和力受体（FcεR）是抗原诱导肥大细胞和嗜碱性粒细胞活化的细胞触发因素。为检测FcεR的功能完整性，我们采用了一种蛋白质植入程序，即将纯化的受体复合物与仙台病毒包膜进行重组。后者促进杂交囊泡与受体细胞如大鼠嗜碱性白血病细胞RBL - 2H3融合，从而作为受体的载体。植入的FcεR与125I标记的小鼠IgE（抗DNP）复合，以实现受体定量以及由DNP20BSA进行特异性触发。在未标记的大鼠IgE存在下进行植入，未标记的大鼠IgE可阻断受体细胞RBL - 2H3上的天然受体，结果导致每10(6)个细胞可掺入多达15 ng受体结合的IgE。这一数量大致相当于此类细胞上天然受体的10 - 20%。由特异性识别植入IgE的试剂在受体细胞中触发的胞吐作用达到最大反应的15%至50%。各种干扰病毒包膜活性的处理会使受体掺入量（降低3 - 5倍）和细胞脱颗粒作用（降低3 - 10倍）均减少。这些处理包括将受体与重组包膜分离、向孵育混合物中添加血清（以抑制囊泡与细胞结合）以及用胰蛋白酶处理病毒（以抑制囊泡与细胞融合）。聚乙二醇8000（4%）可增强受体的掺入及其功能反应。这些处理区分了IgE - FcεR复合物的真正掺入与125I - IgE与受体细胞结合的其他机制。（摘要截短于250词）
