Belcheva M M, Gucker S, Chuang D M, Clark W G, Jefcoat L B, McHale R J, Toth G, Borsodi A, Coscia C J
F.A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri, USA.
J Pharmacol Exp Ther. 1995 Sep;274(3):1513-23.
Opioid binding sites were found in nuclear matrix preparations from NG108-15 neurohybrid cells. Binding parameters of delta-specific radioligands indicated that high-affinity binding sites discovered in purified nuclei were present in nuclear membranes and nuclear matrix fractions. Agonists bind with low affinity, if at all, to nuclear matrix preparations. Neither sensitivity of agonist binding to the GTP analog 5-guanylylimidodiphosphate nor adenylyl cyclase activity were detected in this fraction, suggesting the presence of guanine nucleotide binding regulatory protein/effector uncoupled sites. Opioid inhibition of basal and forskolin-stimulated adenylyl cyclase activity was found in nuclear membrane preparations. Cycloheximide treatment of cells inhibited opioid binding to nuclear membrane fractions to a greater extent than that associated with membranes sedimenting at 20,000 x g (P20) or nuclear matrix. Colchicine, a microtubule disrupter and inhibitor of receptor internalization, caused up-regulation of nuclear membrane and P20 opioid receptors and a loss in nuclear matrix associated sites. Taxol, a microtubule stabilizing agent, prevented the effect of colchicine. Etorphine-elicited down-regulation increased nuclear matrix associated binding while diminishing that in nuclear membranes and P20 fractions. Agonist-induced desensitization completely abolished nuclear matrix binding. In vitro preincubation of nuclear matrix preparations with protein kinase A catalytic subunit mimicked the desensitization effect. Forskolin treatment of cells potentiated nuclear matrix and P20 binding. These data suggest that nuclear membrane opioid receptors represent newly synthesized molecules en route to the cell surface, whereas nuclear matrix contains internalized delta sites.
在NG108 - 15神经杂交细胞的核基质制剂中发现了阿片样物质结合位点。δ特异性放射性配体的结合参数表明，在纯化细胞核中发现的高亲和力结合位点存在于核膜和核基质组分中。激动剂与核基质制剂的结合亲和力很低，甚至根本不结合。在该组分中未检测到激动剂结合对鸟苷三磷酸类似物5 - 鸟苷酰亚胺二磷酸的敏感性，也未检测到腺苷酸环化酶活性，这表明存在鸟嘌呤核苷酸结合调节蛋白/效应器解偶联位点。在核膜制剂中发现了阿片样物质对基础和福斯高林刺激的腺苷酸环化酶活性的抑制作用。用放线菌酮处理细胞对阿片样物质与核膜组分的结合抑制作用，比与在20,000×g沉降的膜（P20）或核基质相关的抑制作用更大。秋水仙碱是一种微管破坏剂和受体内化抑制剂，它导致核膜和P20阿片样物质受体上调，以及核基质相关位点的丧失。紫杉醇是一种微管稳定剂，可阻止秋水仙碱的作用。埃托啡引起的下调增加了核基质相关结合，同时减少了核膜和P20组分中的结合。激动剂诱导的脱敏完全消除了核基质结合。用蛋白激酶A催化亚基对核基质制剂进行体外预孵育模拟了脱敏作用。用福斯高林处理细胞增强了核基质和P20结合。这些数据表明，核膜阿片样物质受体代表了正在运往细胞表面的新合成分子，而核基质含有内化的δ位点。
