Joint Study on Molecular Mechanism of Opioid Receptors

这项联合研究旨在阐明阿片受体分子机制的想法是从凝血酶受体的独特分子特征得出的，在凝血酶受体中，配体多肽是被拴住的。凝血酶受体属于一个具有七个跨膜结构的受体家族，但它是由凝血酶的酶功能激活的，凝血酶可以裂解Arg-41和Ser-42之间的肽键，暴露出一个被拴住的配体SFLLRNP。如果我们构建了含有脑啡肽的阿片受体，这是一种天然的阿片受体的多肽配体，我们可能获得一个新的分子工具来阐明七种跨膜受体的分子机制。在配体系留结合到配体结合部位的过程中，给药的配体将在同一位置与系留配体竞争。例如，当我们使用放射性标记的配体时，这可能允许进行配体解离的速率分析。因此，本研究的目的是建立一种新的构建突变型阿片…的方法最初，内啡肽的N-末端8-肽被掺入δ或μ阿片受体的N-延伸肽中。这种突变的受体包括凝血酶结合位点和一系列酸性氨基酸、内啡肽、裂解位点和标记M1表位，从羧基到氨基的顺序是这样的。虽然脑啡肽类似物可激活该受体，但凝血酶处理不能激活该受体。因此，我们决定利用凝血酶受体本身的N-延伸，它连接到δ阿片受体(其克隆被指定为DOR)。脑啡肽(5个氨基酸，序列为YGGFL)被加入到这个N-延伸中，以取代SFLLRNP。嵌合体受体PADER在COS-7细胞中有表达。表达的受体PADOR被脑啡肽类似物De被激活，尽管它比生物碱配体地丙诺啡弱约10倍。Western blottg分析表明，M1抗体检测到PADOR的构建是完整的，凝血酶裂解N端肽以探索脑啡肽与特异性抗体3E-7的结合。虽然被拴住的脑啡肽对受体的激活程度不大，但我们成功地构建了嵌合体受体，并在其中掺入了多肽配体。因此，这一研究项目的主要目标取得了巨大的科学成就。较少

The idea of this joint study to elucidate the molecular mechanism of opioid receptors has been obtained from the unique molecular feature of thrombin receptor, in which the ligand peptide is tethered. The thrombin receptor is grouped in a family of receptors with seven transmembrane structures, but it is activated by the enzyme function of thrombin that cleaves the peptide bond between Arg-41 and Ser-42 to expose a tethered ligand SFLLRNP. If we construct the opioid receptors containing enkephalin, a natural peptide ligand of d opioid receptor, we may attain a novel molecular tool to clarify the molecular mechanism of seven transmembrane receptors. During the ligand tethered binds to the ligand binding site, the ligand administered would compete with the tethered ligand at the same site. This may allow, for instance, the rate analysis of ligand dissociation when we used a radiolabeled ligand. Thus, the goal of the present study is to establish a novel method to construct a mutant opioi … More d receptor with the tethered ligand peptide in receptor itself.At the beginning N-terminal 8-peptide of endorphin was incorporated in the N-extension peptide of δ or μ opioid receptor. This mutant receptor include the thrombin binding site with a cluster of acidic amino acids, endorphin peptide, cleavage site, and tag M1 epitop, in this order from the carboxyl site to the amino site. Although this receptor was activated by enkephalin analogs administered, no activation was observed by the treatment of thrombin. Thus, we decided to utilize the N-extension of the thrombin receptor per se which was ligated to the δ opioid receptor (the clone of which was designated as dor). Enkephalin (5 amino acids with sequence of YGGFL) was incorporated into this N-extension to replace SFLLRNP. The chimera receptor pador was expressed in the COS-7 cells. Expressed receptor PADOR was found to be activated with enkephalin analog dertorphin II, although it was about ten times weaker than the alkaloid ligand diprenorphin. The Westernblottg analyses indicated that the construction of PADOR is complete as detected by M1 antibody, and that thrombin cleaves the N-terminal peptide to explore enkephalin binding to the specific antibody 3E-7. Although the extent of receptor activation by the tethered enkephalin was not great, we succeeded in the chimera receptor in which peptide ligand is incorporated. Thus, the major goal of this research project was achieved with enormous scientific successes. Less