Specific affinity-labeling of the nociceptin ORL1 receptor using a thiol-activated Cys(Npys)-containing peptide ligand.
Specific affinity-labeling of the nociceptin ORL1 receptor using a thiol-activated Cys(Npys)-containing peptide ligand.
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使用硫醇激活的含 Cys(Npys) 的肽配体对伤害感受素 ORL1 受体进行特异性亲和标记。
DOI：
10.1002/bip.22792
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
and Y. Shimohigashi
中科院分区：
文献类型：
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作者：
A. Matsushima;H. Nishimura;Y. Matsuyama;X. Liu;T. Costa;and Y. Shimohigashi
我们之前发现了一种基于拮抗剂的肽配体，H‐Cys（Npys）‐Arg‐Tyr‐Tyr‐Arg‐Ile‐Lys‐NH2，可以捕获痛感肽受体ORL1配体结合位点的游离巯基。然而，这种硫醇-二硫交换反应的确切受体位点尚未被发现，尽管这种鉴定将有助于澄清配体识别位点。由于Cys→Ala取代阻止了反应，我们对ORL1受体跨膜（TM）结构域中的所有Cys残基进行了所谓的Ala扫描。七种不同的突变受体在COS‐7细胞中完全表达，并通过使用nociceptin和[3H]nociceptin的竞争结合试验检测其特异性亲和力标记。体外扫描结果显示，TM1中的标记残基为Cys59， TM5中的标记残基为Cys215和Cys231， TM7中的标记残基为Cys310。本研究提供了一种新的Cys（Npys）亲和标记方法，用于鉴定ORL1受体的配体结合位点。©2016 Wiley期刊公司生物工程学报（自然科学版），2016,35(6):464 - 467。
We previously showed that an antagonist‐based peptide ligand, H‐Cys(Npys)‐Arg‐Tyr‐Tyr‐Arg‐ Ile‐Lys‐NH2, captures the free thiol groups in the ligand‐binding site of the nociceptin receptor ORL1. However, the exact receptor sites of this thiol‐disulfide exchange reaction have not been uncovered, although such identification would help to clarify the ligand recognition site. Since the Cys→Ala substitution prevents the reaction, we performed the so‐called Ala scanning for all the Cys residues in the transmembrane (TM) domains of the ORL1 receptor. Seven different mutant receptors were soundly expressed in the COS‐7 cells and examined for their specific affinity labeling by a competitive binding assay using nociceptin and [3H]nociceptin. The results ofin vitroAla scanning analyses revealed that the labeled residues were Cys59 in TM1, Cys215 and Cys231 in TM5, and Cys310 in TM7. The present study has provided a novel method of Cys(Npys)‐affinity labeling for identification of the ligand‐binding sites in the ORL1 receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 460–469, 2016.