Ko J L, Lee N M, Loh H H
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455.
J Biol Chem. 1992 Jun 25;267(18):12722-7.
Cross-linking of beta-125I-endorphin to NG108-15 cell membranes labeled bands with molecular masses of 55, 35, and 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We applied several criteria to evaluate the relevance of these cross-linked bands to delta-opioid receptors, including selectivity, stereospecificity, affinity, G-protein coupling, down-regulation, and correlation with opioid receptor level in different well-characterized cell lines. Only the 25 kDa protein adequately fulfilled all these criteria. Thus, cross-linking to the 25-kDa band was selectively inhibited by ligands with delta-opioid affinity, but not by mu-opioid, kappa-opioid, or optically inactive opioid ligands or by non-opioid ligands. Based on inhibition of cross-linking, we calculated an affinity of [D-Ala2,D-Leu5]enkephalin binding to the 25-kDa and (Kd = 6 nM) that is similar to that reported for [D-Ala2,D-Leu5]enkephalin binding to NG108-15 membranes; this affinity decreased approximately 10-fold in the presence of Na+/guanyl-5'-yl imidodiphosphate. Chronic agonist treatment of NG108-15 cells reduced cross-linking to the 25-kDa band, but not to others, in a manner parallel to down-regulation of opioid receptors. Finally, the amount of the 25-kDa band was roughly proportional to the level of opioid receptors present in N18TG2, NS20Y, ST7-3, and ST8-4 cells. The 25-kDa band was absent in PC12h, NIH3T3, and C6BU1 cells as well as in liver, all of which had no detectable opioid binding.
β-125I-内啡肽与NG108-15细胞膜交联后，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上标记出分子量为55、35和25 kDa的条带。我们应用了几个标准来评估这些交联条带与δ-阿片受体的相关性，包括选择性、立体特异性、亲和力、G蛋白偶联、下调以及与不同特征明确的细胞系中阿片受体水平的相关性。只有25 kDa的蛋白充分满足了所有这些标准。因此，与δ-阿片亲和力的配体可选择性抑制与25 kDa条带的交联，但μ-阿片、κ-阿片或光学无活性的阿片配体或非阿片配体则不能。基于交联抑制，我们计算出[D-Ala2,D-Leu5]脑啡肽与25 kDa条带的结合亲和力（Kd = 6 nM），这与报道的[D-Ala2,D-Leu5]脑啡肽与NG108-15细胞膜的结合亲和力相似；在存在Na+/鸟苷-5'-基亚氨基二磷酸的情况下，这种亲和力降低了约10倍。对NG108-15细胞进行慢性激动剂处理可减少与25 kDa条带的交联，但不影响其他条带，其方式与阿片受体下调平行。最后，25 kDa条带的量大致与N18TG2、NS20Y、ST7-3和ST8-4细胞中存在的阿片受体水平成正比。PC12h、NIH3T3和C6BU1细胞以及肝脏中均不存在25 kDa条带，所有这些细胞均未检测到阿片结合。
