Joint Study on Molecular Mechanism of Opioid Receptors

本联合研究阐明阿片受体分子机制的思路，是从凝血酶受体独特的配体肽系链的分子特征中获得的。凝血酶受体被归为具有7个跨膜结构的受体家族，但它被凝血酶的酶功能激活，该酶功能可切割Arg-41和Ser-42之间的肽键，以暴露栓系配体SFLLRNP。如果我们构建含有脑啡肽（d -阿片受体的天然肽配体）的阿片受体，我们可能获得一种新的分子工具来阐明7种跨膜受体的分子机制。在配体与配体结合位点结合的过程中，所给配体会在同一位点与所给配体竞争。这可能允许，例如，当我们使用放射性标记配体时，配体解离的速率分析。因此，本研究的目的是建立一种新的方法来构建具有拴链配体肽的阿片受体突变体。起初，内啡肽的n端8肽结合在δ或μ阿片受体的n延伸肽中。该突变受体包括凝血酶与一簇酸性氨基酸结合位点、内啡肽肽、裂解位点和标签M1表位，顺序从羧基位点到氨基位点。虽然这种受体被脑啡肽类似物激活，但没有被凝血酶激活。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（Enkephalin）（具有YGGFL序列的5个氨基酸）被纳入该N-extension以取代SFLLRNP。嵌合体受体pador在COS-7细胞中表达。表达的受体PADOR被脑啡肽类似物dertorphin II激活，尽管它比生物碱配体diprenorphin弱约10倍。Westernblottg分析表明，M1抗体检测到PADOR的构建完成，凝血酶切割n端肽，探索脑啡肽与特异性抗体3E-7的结合。虽然栓系脑啡肽对受体的激活程度并不大，但我们成功地获得了肽类配体掺入的嵌合体受体。因此，这项研究项目的主要目标取得了巨大的科学成就。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