| Co-Investigator(Kenkyū-buntansha) |
FUJITA Tsugumi Graduate School of Science, Kyushu University, DC3 (Research Assistant), 大学院・理学研究科, 学振特別研究員
中馬 吉郎 九州大学, 大学院・理学研究科, 学振特別研究員
NOSE Takeru Graduate School of Science, Kyushu University, Research Associate, 大学院・理学研究科, 助手 (10301334)
CHUMAN Yoshihiro Graduate School of Science, Kyushu University, DC2 (Research Assistant)
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| Research Abstract |
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
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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
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