Direct identification of human oxytocin receptor-binding domains using a photoactivatable cyclic peptide antagonist -: Comparison with the human V1a vasopressin receptor
Direct identification of human oxytocin receptor-binding domains using a photoactivatable cyclic peptide antagonist -: Comparison with the human V1a vasopressin receptor
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DOI：
10.1074/jbc.m102073200
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发表时间：
2001-07-20
影响因子：
4.8
通讯作者：
Mouillac, B
中科院分区：
文献类型：
--
作者：
Breton, C;Chellil, H;Mouillac, B
了解负责拮抗剂结合催产素受体的分子决定因素，应提供重要的见解，促进合理设计潜在的治疗药物治疗早产。为了研究配体/受体的相互作用，我们使用了一种新型的光敏放射性碘标记的人催产素受体拮抗剂d（CH 2）（5）[Tyr（Me）（2），Thr（4），Orn（8），Phe（3（125）I，4 N（3））-NH 29]vasotocin。该配体对中国仓鼠卵巢细胞中表达的人催产素和V-1a加压素受体具有相当高的亲和力。利用这种双重特异性，我们对两种受体进行了光亲和标记实验，光标记的催产素和V-1a受体在70-75 kDa处出现一条独特的蛋白带，在85-90和46 kDa处分别出现两条标记蛋白带。为了鉴定拮抗剂和受体之间的接触位点，标记的70-75-和46-kDa蛋白用CNBr切割并用Lys-C和Arg-C内蛋白酶消化。片段化模式允许鉴定催产素受体跨膜结构域III中由残基Leu（114）-瓦尔（115）- Lys（116）组成的共价标记区域。对V-1a受体中接触位点的分析导致鉴定出由残基瓦尔（126）-瓦尔（127）-Lys（128）组成的同源区。结合域被证实的突变的几个CNBr切割位点的催产素受体和一个Lys-C切割位点的V-1a受体。结果与以前的实验数据和三维模型的激动剂和拮抗剂结合的催产素/血管加压素受体家族的成员。
Understanding of the molecular determinants responsible for antagonist binding to the oxytocin receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand/receptor interactions, we used a novel photosensitive radioiodinated antagonist of the human oxytocin receptor, d(CH2)(5) [Tyr(Me)(2),Thr(4),Orn(8),Phe(3(125)I,4N(3))-NH29]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V-1a vasopressin receptors expressed in Chinese hamster ovary cells. Taking advantage of this dual specificity, we conducted photoaffinity labeling experiments on both receptors, Photolabeled oxytocin and V-1a receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at 85-90 and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70-75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases, The fragmentation patterns allowed the identification of a covalently labeled region in the oxytocin receptor transmembrane domain III consisting of the residues Leu(114)-Val(115)- Lys(116). Analysis of contact sites in the V-1a receptor led to the identification of the homologous region consisting of the residues Val(126)-Val(127)-Lys(128). Binding domains were confirmed by mutation of several CNBr cleavage sites in the oxytocin receptor and of one Lys-C cleavage site in the V-1a receptor. The results are in agreement with previous experimental data and three-dimensional models of agonist and antagonist binding to members of the oxytocin/vasopressin receptor family.