Afify E A, Law P Y, Riedl M, Elde R, Loh H H
Department of Pharmacology, Medical School, University of Minnesota, Minneapolis 55455, USA.
Brain Res Mol Brain Res. 1998 Feb;54(1):24-34. doi: 10.1016/s0169-328x(97)00315-x.
Chronic exposure of mu-and delta-opioid receptors to their agonists leads to different rates in receptor down-regulation. In order to analyze the role of the carboxyl terminus of mu-and delta-opioid receptors in the difference in the rate of down-regulation, two chimeras of these receptors were generated by swapping the carboxyl termini; MORTAGDT and DORTAGMT. These chimeras were tagged at the N-terminus with hemagglutinin (HA) epitope (YPYDVPDYA), which can be recognized by the monoclonal antibody 12CA5, and then stably expressed in Neuro 2A (N2A) cells. The swapping of the carboxyl termini did not alter the ligand selectivity of these receptor chimeras. However, they did exhibit a reduction in agonist potency to inhibit forskolin-stimulated adenylyl cyclase activity for all agonists tested except etorphine which had a potency comparable to that of wild type receptors. Treatment of the N2A cells expressing MORTAGDT with 50 nM etorphine produced a faster rate of receptor down-regulation when compared to the wild type mu-opioid receptor. Immunofluorescence microscopy of the MORTAGDT chimera using a monoclonal antibody against HA confirmed internalization of the receptors after treatment with etorphine for 1 and 6h. There was a reduction in the HA-immunoreactivity at the cell surface of the MORTAGDT chimera concurrent with more noticeable HA-immunoreactivity inside the cell compared to the wild type receptor. On the other hand, the rate of down-regulation of DORTAGMT receptors was seen to be the same as the wild type delta-opioid receptor after etorphine treatment. Immunofluorescence studies showed more reduction in cell surface staining of the DORTAGMT chimera compared to the wild type receptor. These data suggest the involvement of the carboxyl terminus in agonist-induced down-regulation and internalization of the nu-opioid receptor. However, different mechanisms that are unrelated to the carboxyl terminus may operate in the down-regulation of delta-opioid receptor.
μ-阿片受体和δ-阿片受体长期暴露于其激动剂会导致不同程度的受体下调。为了分析μ-阿片受体和δ-阿片受体的羧基末端在下调速率差异中的作用，通过交换羧基末端产生了这两种受体的两个嵌合体；MORTAGDT和DORTAGMT。这些嵌合体在N端用可被单克隆抗体12CA5识别的血凝素（HA）表位（YPYDVPDYA）进行标记，然后在Neuro 2A（N2A）细胞中稳定表达。羧基末端的交换并未改变这些受体嵌合体的配体选择性。然而，除埃托啡外，对于所有测试的激动剂，它们抑制福斯高林刺激的腺苷酸环化酶活性的激动剂效力均有所降低，而埃托啡的效力与野生型受体相当。与野生型μ-阿片受体相比，用50 nM埃托啡处理表达MORTAGDT的N2A细胞时，受体下调速率更快。使用抗HA单克隆抗体对MORTAGDT嵌合体进行免疫荧光显微镜检查证实，用埃托啡处理1小时和6小时后，受体会发生内化。与野生型受体相比，MORTAGDT嵌合体细胞表面的HA免疫反应性降低，同时细胞内的HA免疫反应性更明显。另一方面，埃托啡处理后，DORTAGMT受体的下调速率与野生型δ-阿片受体相同。免疫荧光研究表明，与野生型受体相比，DORTAGMT嵌合体的细胞表面染色减少得更多。这些数据表明羧基末端参与了激动剂诱导的μ-阿片受体下调和内化。然而，与羧基末端无关的不同机制可能在δ-阿片受体的下调中起作用。
