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首页> 外文期刊>Canadian Journal of Physiology and Pharmacology >Preliminary mutational analysis of the human kinin B2 receptor for nonpeptide antagonist ligands recognition.
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Preliminary mutational analysis of the human kinin B2 receptor for nonpeptide antagonist ligands recognition.

机译：人类激肽B2受体对非肽拮抗剂配体识别的初步突变分析。

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摘要

FR173657, LF16,0335, and LF16,0687 are nonpeptide antagonists, endowed with high affinity and selectivity for the human kinin B2 receptor. The kinin B2 receptor belongs to the family of G-protein-coupled receptors with seven transmembrane (TM) helices. In the present study, we aimed, through computer-assisted modeling and mutagenesis, to identify residues in the human B2 receptor (hB2R) amino acid sequence that are involved in nonpeptide antagonist binding in order to build up experimental data as a first step towards a molecular model of nonpeptide ligands binding site. Fourteen amino acid residues within the TM segments were mutated to alanine. The wild type and mutant receptors were stably expressed in Chinese hamster ovary (dhfr-) cells and tested for their ability to bind agonist ([3H]bradykinin) and peptide antagonist ([3H]MENI 1270) radioligands. The affinity of nonpeptide ligands was determined by heterologous competition experiments using the above radioligands. We found that some mutations in TM2 (W86A) and TM7 (Y295A, N297A) impair the binding affinity of the three nonpeptide antagonists. On the other hand, some mutated residues in TM3 (S1 17A) and TM6 (W256A) reduce the affinity of LF16,0335 and LF16,0687 only. Results are discussed in order to build up a hypothesis for the likely different interactions of various nonpeptide ligands with the B2 receptor.
机译：FR173657，LF16,0335和LF16,0687是非肽类拮抗剂，对人激肽B2受体具有高亲和力和选择性。激肽B2受体属于具有七个跨膜（TM）螺旋的G蛋白偶联受体家族。在本研究中，我们旨在通过计算机辅助建模和诱变来鉴定人B2受体（hB2R）氨基酸序列中与非肽拮抗剂结合所涉及的残基，以建立实验数据，作为迈向非肽配体结合位点的分子模型。 TM区段内的十四个氨基酸残基突变为丙氨酸。野生型和突变受体在中国仓鼠卵巢（dhfr-）细胞中稳定表达，并测试其与激动剂（[3H]缓激肽）和肽拮抗剂（[3H] MENI 1270）放射配体结合的能力。使用上述放射性配体通过异源竞争实验确定非肽配体的亲和力。我们发现，TM2（W86A）和TM7（Y295A，N297A）中的某些突变会损害三种非肽拮抗剂的结合亲和力。另一方面，TM3（S1 17A）和TM6（W256A）中的某些突变残基仅降低LF16,0335和LF16,0687的亲和力。为了建立各种非肽配体与B2受体可能不同相互作用的假设，对结果进行了讨论。

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