The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan [b]chromen-1α-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors
The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan [b]chromen-1α-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors
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DOI：
10.1074/jbc.m006230200
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发表时间：
2000-10-27
影响因子：
4.8
通讯作者：
Kuhn, R
中科院分区：
文献类型：
--
作者：
Pagano, A;Rüegg, D;Kuhn, R
我们研究了一种新的人代谢型谷氨酸受体5（hmGluR 5）拮抗剂2-甲基-6-（苯乙炔基）吡啶（2-methyl-6-（phenylethynyl）pyridine，MPEP）的抑制机制和作用部位。MPEP还在很大程度上抑制瞬时过表达大鼠mGluR 5的细胞中的组成型受体活性，表明MPEP充当反向激动剂。为了研究控制选择性配体结合的分子决定簇，使用hmGluR 1和hmGluR 5的嵌合体和单个氨基酸取代进行诱变研究，测试突变体与新型mGluR 5放射性配体[H-3]2-甲基-6-甲基-N-（2-甲基-N-（3-甲氧基苯基）乙炔基吡啶（M-MPEP），MPEP的类似物，用hmGluR 1的同源残基取代跨膜（TM）VII中的Ala-810或TMIII中的Pro-655和Ser-658，可使放射性配体与[H-3]M-MPEP的结合消失。相反，带有hmGluR 5的这三个残基的hmGluR 1反向突变体显示出对[H-3]M-MPEP的高亲和力。7-羟基亚氨基环丙烷[B]色烯-1a-羧酸乙酯也能抑制放射性配体与这些突变体的结合（CPC-COEt），一种结构上不相关的非竞争性mGluR 1拮抗剂，先前显示与hmGluR 1的TMVII中的残基Thr-815和Ala-818相互作用，这些结果表明，MPEP和CPCCOEt与I组mGluRs的TBI区域中的重叠结合口袋结合，但与不同的非结合口袋相互作用。保守残基
We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands, Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist, To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5, The mutants were tested for binding of the novel mGluR5 radioligand [H-3]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP, Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding, In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [H-3]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPC-COEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1, These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TBI region of group I mGluRs but interact with different non conserved residues.