Chimeric NK1 (substance P)/NK3 (neurokinin B) receptors. Identification of domains determining the binding specificity of tachykinin agonists.
Chimeric NK1 (substance P)/NK3 (neurokinin B) receptors. Identification of domains determining the binding specificity of tachykinin agonists.
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DOI：
10.1016/s0021-9258(18)53041-6
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发表时间：
1993-04
期刊：
影响因子：
--
通讯作者：
U. Gether;T. Johansen;T. Schwartz
中科院分区：
文献类型：
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作者：
U. Gether;T. Johansen;T. Schwartz
NK1（P 物质）和 NK3（神经激肽 B）受体是 G 蛋白偶联受体，在跨膜域内具有大约 70% 的同一性。然而，它们各自在肽结合方面具有独特的药理学特征。为了鉴定决定其对天然和合成速激肽的选择性的表位，我们构建了一系列嵌合NK1/NK3受体，其中NK1受体中长度增加的羧基末端片段与NK3受体中的相应片段交换。嵌合构建体的总体结构完整性通过两栖动物速激肽肽、eleoisin 得到证实，两种野生型受体均能很好地识别该肽，并以相似甚至更高的亲和力与所有嵌合受体结合。竞争结合研究表明，两种天然配体P物质和神经激肽B的亲和力通过一系列嵌合受体逐渐改变，表明整个受体结构中的几个结合表位参与了这些肽的选择性识别。然而，当受体氨基末端的片段交换时，对 P 物质的结合亲和力的单一且最大的变化发生，而神经激肽 B 则发生在受体的羧基末端。 NK1 选择性配体 [Sar9,Met(O2)11]SP 的亲和力在一系列嵌合受体中的变化甚至比 P 物质的亲和力变化更缓慢。相反，NK3 选择性六肽 Senktide 仅当 NK3 受体的跨膜片段 III 和 IV 并入嵌合构建体时才被识别。这些数据表明，几个受体结构域有助于速激肽激动剂的结合特异性，但对每种肽的影响程度不同。结论是速激肽以部分不同的方式与分散在整个受体结构中的多个表位相互作用，但可以想象这些表位紧密地位于假设的受体中心周围的空间中。
The NK1 (substance P) and NK3 (neurokinin B) receptors are G protein-coupled receptors sharing approximately 70% identity within the membrane-spanning domains. However, they each have a distinct pharmacological profile in respect of peptide binding. To identify epitopes that determine their selectivity for natural and synthetic tachykinin peptides, we constructed a series of chimeric NK1/NK3 receptors in which carboxyl-terminal segments of increasing length in the NK1 receptor were exchanged with the corresponding segments from the NK3 receptor. The general, structural integrity of the chimeric constructs was confirmed by the amphibian tachykinin peptide, eledoisin, which was recognized equally well by both of the wild-type receptors and bound with a similar or even higher affinity to all the chimeric receptors. Competition binding studies showed that the affinity of the two natural ligands, substance P and neurokinin B, changed gradually through the series of chimeric receptors indicating that several binding epitopes throughout the receptor structure are involved in the selective recognition of these peptides. However, whereas the single, largest change in binding affinity for substance P occurred when segments in the amino-terminal end of the receptor were exchanged, this occurred for neurokinin B in the carboxyl-terminal end of the receptor. The affinity of the NK1-selective ligand, [Sar9,Met(O2)11]SP, changed even more gradually through the series of chimeric receptors than that of substance P. In contrast, the NK3-selective hexapeptide, senktide, was recognized only when transmembrane segment III and IV from the NK3 receptor were incorporated into the chimeric constructs. These data suggest that several receptor domains contribute to the binding specificity of tachykinin agonists but in varying degrees for each peptide. It is concluded that the tachykinin peptides, in partially different ways, interact with multiple epitopes scattered throughout the receptor structure, but conceivably these epitopes are closely located in space around a hypothetical receptor center.