Cascieri M A, Huang R R, Fong T M, Cheung A H, Sadowski S, Ber E, Strader C D
Department of Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065.
Mol Pharmacol. 1992 Jun;41(6):1096-9.
We have measured the affinity of various analogs and fragments of the tachykinin substance P for the cloned rat NK1, NK2, and NK3 receptors heterologously expressed in Chinese hamster ovary cells. The hydrophobic carboxyl-terminal pentapeptide sequence substance P-(7-11) binds with similar affinity (2-20 microM) to all three receptors. Our data suggest that addition of one to three amino-terminal residues to this sequence results in the optimization of its interaction within the binding pocket of the NK1 receptor. The addition of Pro-Gln-Gln to the carboxyl-terminal pentapeptide sequence increases affinity for the NK1 receptor, either by providing additional binding interactions or by modifying the conformation of the carboxyl-terminal sequence. This latter hypothesis is supported by the observation that physalaemin and phyllomedusin, which also contain a proline residue in the position analogous to the proline residue 4 of substance P, are also selective for NK1 receptors. Tachykinins that lack this proline have no higher affinity for NK1 than [pGlu] substance P-(6-11). Conversely, addition of Pro-Gln-Gln to the carboxyl-terminal pentapeptide sequence is unfavorable for NK2 and NK3 receptor binding. Preliminary data suggest that tachykinins with high affinity (Kd less than 500 nM) for NK2 receptors contain an aspartate residue in the position analogous to residue 5 of substance P, suggesting that an ionic interaction with the receptor may contribute binding energy. Further experiments will be required to determine the structural determinants of the NK1, NK2, and NK3 receptors responsible for these binding properties.
我们已测定了速激肽P物质的各种类似物和片段对在中国仓鼠卵巢细胞中异源表达的克隆大鼠NK1、NK2和NK3受体的亲和力。疏水性的羧基末端五肽序列P物质-(7-11) 与所有三种受体的结合亲和力相似（2-20微摩尔）。我们的数据表明，在该序列上添加一至三个氨基末端残基可优化其在NK1受体结合口袋内的相互作用。在羧基末端五肽序列上添加Pro-Gln-Gln可增加对NK1受体的亲和力，这可能是通过提供额外的结合相互作用或改变羧基末端序列的构象来实现的。后一种假设得到了以下观察结果的支持：physalaemin和phyllomedusin在与P物质的脯氨酸残基4类似的位置也含有一个脯氨酸残基，它们也对NK1受体具有选择性。缺乏这种脯氨酸的速激肽对NK1的亲和力并不比[pGlu]P物质-(6-11)更高。相反，在羧基末端五肽序列上添加Pro-Gln-Gln不利于与NK2和NK3受体结合。初步数据表明，对NK2受体具有高亲和力（解离常数小于500纳摩尔）的速激肽在与P物质的残基5类似的位置含有一个天冬氨酸残基，这表明与受体的离子相互作用可能有助于结合能。需要进一步的实验来确定负责这些结合特性的NK1、NK2和NK3受体的结构决定因素。
