Varga E V, Li X, Stropova D, Zalewska T, Landsman R S, Knapp R J, Malatynska E, Kawai K, Mizusura A, Nagase H, Calderon S N, Rice K, Hruby V J, Roeske W R, Yamamura H I
Department of Pharmacology, The University of Arizona, College of Medicine, Tucson 85724, USA.
Mol Pharmacol. 1996 Dec;50(6):1619-24.
In the present study, we replaced the third extracellular loop of the human delta-opioid receptor with that of the human mu-opioid receptor. A modified polymerase chain reaction overlap extension method was used to achieve the exact splicing in the chimera to show the importance of the extracellular loop in ligand binding without interference from transmembrane substitutions. The replacement of the third extracellular loop did not alter the affinity of [3H]diprenorphine but caused a dramatic decrease in the affinity of both the delta-selective peptide agonists cyclo[D-Pen2,4'Cl-Phe4,D-Pen5]enkephalin and deltorphin II and the delta-selective nonpeptide agonists SNC 121 and (-)TAN 67. The affinities of the mu-selective peptide agonist [D-Ala2-MePhe4-Gly-ol5]enkephalin and the mu-preferring nonpeptide agonist morphine were not affected. Site-directed mutagenesis studies show that the mechanism of ligand recognition might be different for each structural class of opioid ligands.
在本研究中，我们用人μ-阿片受体的第三个细胞外环替换了人δ-阿片受体的相应环。采用改良的聚合酶链反应重叠延伸法在嵌合体中实现精确拼接，以显示细胞外环在配体结合中的重要性，而不受跨膜取代的干扰。第三个细胞外环的替换并未改变[³H]二丙诺啡的亲和力，但导致δ-选择性肽激动剂环[D-青霉胺²，4'-氯苯丙氨酸⁴，D-青霉胺⁵]脑啡肽和强啡肽II以及δ-选择性非肽激动剂SNC 121和（-）TAN 67的亲和力显著降低。μ-选择性肽激动剂[D-丙氨酸²-甲基苯丙氨酸⁴-甘醇⁵]脑啡肽和偏向μ型的非肽激动剂吗啡的亲和力未受影响。定点诱变研究表明，阿片类配体的每种结构类型的配体识别机制可能不同。
