Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, and Risk Science Research Center, Kyushu University, Fukuoka 812-8581, Japan.
Bioorg Med Chem. 2011 Dec 15;19(24):7597-602. doi: 10.1016/j.bmc.2011.10.024. Epub 2011 Oct 17.
All of the δ, μ, and κ opioid receptors have a free thiol group of the Cys residue in the ligand-binding site, although its functional role is not yet known. In order to examine whether or not a similar Cys is also present in the ORL1 nociceptin receptor, we attempted to identify it by affinity labeling using a specific antagonist peptide. We first treated ORL1-expressing COS-7 cell membrane preparations with the thiol-alkylation reagent N-ethylmaleimide (NEM) to perform a binding assay using [(3)H]nociceptin as a tracer and nociceptin, an ORL1 agonist, or Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2), a nociceptin/ORL1 antagonist, as a competitor. It was suggested that ORL1 has a free Cys in its ligand-binding site, since the NEM treatment reduced the population of ligand-binding sites. This was further confirmed by affinity labeling using Cys(Npys)-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) with the SNpys group that can react with a free thiol group, resulting in the formation of a disulfide bond. This affinity labeling was approximately 23 times more specific than NEM alkylation. The results revealed that the ORL1 nociceptin receptor does contain a free Cys residue in the ligand-binding site.
所有的 δ、μ 和 κ 阿片受体在配体结合部位都有一个 Cys 残基的游离巯基，尽管其功能作用尚不清楚。为了研究 ORL1 孤啡肽受体是否也存在类似的 Cys，我们尝试通过使用特异性拮抗剂肽进行亲和标记来鉴定它。我们首先用巯基烷化试剂 N-乙基马来酰亚胺（NEM）处理表达 ORL1 的 COS-7 细胞膜制剂，并用 [(3)H]孤啡肽作为示踪剂进行结合测定，用孤啡肽（ORL1 激动剂）或 Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2)（孤啡肽/ORL1 拮抗剂）作为竞争剂。由于 NEM 处理降低了配体结合位点的数量，因此表明 ORL1 在其配体结合部位具有游离的 Cys。这进一步通过使用 SNpys 基团与游离巯基反应形成二硫键的 Cys(Npys)-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2)进行亲和标记得到证实。这种亲和标记比 NEM 烷化作用特异性高约 23 倍。结果表明，ORL1 孤啡肽受体确实在配体结合部位含有一个游离的 Cys 残基。
