Meng F, Ueda Y, Hoversten M T, Taylor L P, Reinscheid R K, Monsma F J, Watson S J, Civelli O, Akil H
Mental Health Research Institute, University of Michigan, Ann Arbor 48109, USA.
Mol Pharmacol. 1998 Apr;53(4):772-7. doi: 10.1124/mol.53.4.772.
Although much has been learned about the mechanisms of ligand selectivity between different opioid receptor subtypes, little is known about the common opioid binding pocket shared by all opioid receptors. The recently discovered orphanin system offers a good opportunity to study the mechanisms involved in the binding of opioid versus nonopioid ligands. In the current study, we adopt a "gain of function" approach aimed at shifting the binding profile of the orphanin FQ receptor toward that of the opioid receptors. After two rounds of mutagenesis, several orphanin FQ receptor mutants can be labeled with the opiate alkaloid [3H]naltrindole and show greatly increased affinities toward the opiate antagonists naltrexone, nor-binaltrophine HCl, and (-)-bremazocine. These orphanin FQ receptor mutants also display stereospecificity similar to that of opioid receptors. Furthermore, the orphanin FQ receptor mutant that has the best affinities toward the opioid alkaloids shows, in the presence of GTP and high salt concentration, an affinity-shift profile similar to that of the delta receptor. Most strikingly, the same mutant exhibits naltrindole-sensitive etorphine-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding, whereas the effect of etorphine on GTP binding cannot be inhibited by naltrindole in the wild-type receptor. Our results indicate that 1) several residues in the orphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists; and 2) mutating these residues to those of the opioid receptor at the corresponding position preserves the agonist/antagonist nature of opiate alkaloids as they interact with the mutant receptor. It is reasonable to hypothesize that the corresponding residues in the opioid receptors may form a functional common binding pocket for opiate alkaloids. These findings may be helpful to medicinal chemists in designing ligands for the orphanin FQ receptor based on the structure of the opiate alkaloids.
尽管在不同阿片受体亚型之间配体选择性的机制方面已经有了很多了解，但对于所有阿片受体共有的阿片结合口袋却知之甚少。最近发现的孤啡肽系统为研究阿片类与非阿片类配体结合所涉及的机制提供了一个很好的机会。在当前的研究中，我们采用了一种“功能获得”方法，旨在使孤啡肽FQ受体的结合特征向阿片受体的结合特征转变。经过两轮诱变后，几个孤啡肽FQ受体突变体能够被阿片生物碱[3H]纳曲吲哚标记，并对阿片拮抗剂纳曲酮、盐酸去甲二氢吗啡酮和(-)-布马佐辛表现出大大增加的亲和力。这些孤啡肽FQ受体突变体还表现出与阿片受体相似的立体特异性。此外，对阿片生物碱具有最佳亲和力的孤啡肽FQ受体突变体在存在GTP和高盐浓度的情况下，表现出与δ受体相似的亲和力转变特征。最引人注目的是，相同的突变体表现出纳曲吲哚敏感的埃托啡刺激的[35S]鸟苷-5'-O-(3-硫代)三磷酸结合，而在野生型受体中，埃托啡对GTP结合的作用不能被纳曲吲哚抑制。我们的结果表明：1)孤啡肽FQ受体中的几个残基对其对阿片生物碱尤其是拮抗剂的选择性至关重要；2)将这些残基突变为阿片受体相应位置的残基可保持阿片生物碱与突变受体相互作用时的激动剂/拮抗剂性质。有理由推测，阿片受体中的相应残基可能形成阿片生物碱的功能性共同结合口袋。这些发现可能有助于药物化学家基于阿片生物碱的结构设计孤啡肽FQ受体的配体。
