亮氨酸-2-丙氨酸脑啡肽诱导在中国仓鼠卵巢细胞中稳定表达的δ阿片受体内吞(英文)

Leucine-2-alanine enkephalin induced δ opioid receptors internalization expressed stably in CHO cells

研究在中国仓鼠卵巢（ＣＨＯ）细胞中稳定表达的δ阿片受体（ＤＯＲ）内吞现象及其Ｃ－末端在受体内吞中的作用．方法：用免疫荧光共聚焦显微镜显示受体的着膜；以耐酸性缓冲液洗涤法测定放射性配体－受体结合量确定受体内吞程度．结果：表达的野生（ＣＨＯ－Ｗ）和Ｃ－末端截短（ＣＨＯ－Ｔ）的ＤＯＲ都被正确转运到细胞膜上，激动剂［３Ｈ］亮氨酸－２－丙氨酸脑啡肽（ＬＡＥ）孵育诱导ＤＯＲ快速内吞，拮抗剂［３Ｈ］二丙诺啡（Ｄｉｐ）则不能；ＤＯＲ内吞对胞外高渗透压和温度敏感，但不受百日咳毒素和广谱蛋白激酶抑制剂星形孢菌素的影响；Ｃ－末端截短的ＤＯＲ在ＣＨＯ－Ｔ中的最大内吞不变，但增加较缓慢，结论：ＤＯＲ内吞是一种衣被小泡和激动剂依赖的过程，但不需受体后信号传导．Ｃ－末端与受体的胞膜锚定无关，但可影响受体内吞的初始化。

AIM: To characterize the internalization of δ opioid receptors (DOR) stably expressed in Chinese baluster ovary (CHO) cells and the role of the C-terminal in this process. METHODS: Receptor membrane anchoring was shown by immunofluorescence micro scopy. Receptor internalization was assessed by measuring the radioligand binding resistant to the acidbuffer wash. RESULTS: Originally, all the wildtype (CHO-W) and C-truncated (CHO-T) DOR expressed were localized to the membrane. Agonist [3H] leucine-2-alpine enkephalin (LAE) but not the antagonist [3H]diprenorphine (Dip) induced rapid receptor internalization. The intemalization of Ctruncated DOR in CHO-T was similar to that of the wild-type in maximal level, but climbed up more slowly. DOR internalization was extracellular osmolarity- and temperature-sensitive. Pertussis toxin and universal protein kinase inhibitor staurosporine had no effect on it. CONCLUSION: DOR internalization is an agonist and clathrin-coated pits dependent, but post-receptor cellular signal transduction independent process; moreover, the C-terminal of DOR, not engaged in membrane anchoring, affects the initialization of DOR internalization.