Characterization of the kainate-binding domain of the glutamate receptor GluR-6 subunit
Characterization of the kainate-binding domain of the glutamate receptor GluR-6 subunit
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DOI：
10.1042/bj3301461
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发表时间：
1998-03-15
影响因子：
4.1
通讯作者：
Kuusinen, A
中科院分区：
文献类型：
--
作者：
Keinänen, K;Jouppila, A;Kuusinen, A
红藻氨酸选择性谷氨酸受体亚基GluR-6的重组片段在昆虫细胞中表达，并分析[H-3]红藻氨酸结合活性，以表征负责配体识别的结构决定因素。N-末端缺失约400个氨基酸残基，C-末端缺失约90个氨基酸残基，得到一个膜结合的核心片段，该片段显示出类似于[H-3]红藻氨酸的结合特性。用亲水性接头肽进一步替换膜包埋的区段M1-M3产生可溶性多肽，其在培养基中积累。当通过C-末端组氨酸标签与螯合琼脂糖凝胶珠结合时，可溶性片段显示[H-3]红藻氨酸的低亲和力结合，其以浓度依赖性方式被未标记的软骨藻酸、L-谷氨酸和6-氰基-7-硝基喹喔啉-2，3-二酮取代。我们的研究结果表明，红藻氨酸结合位点是专门由两个不连续的胞外片段（S1和S2），这是同源的细菌氨基酸结合蛋白。GluR-6和GluR-D亚基之间可溶性S1-S2嵌合体的配体结合特征表明，尽管S1和S2片段都有助于激动剂选择性，但GluR-D S2片段的N-末端三分之一足以赋予嵌合体配体结合结构域α-氨基-3-羟基-5-甲基-4-异恶唑丙酸酯结合能力。
Recombinant fragments of the kainate-selective glutamate receptor subunit GluR-6 were expressed in insect cells and analysed for [H-3]kainate binding activity in order to characterize the structural determinants responsible for ligand recognition. Deletion of the N-terminal similar to 400 amino-acid-residue segment and the C-terminal similar to 90 residues resulted in a membrane-bound core fragment which displayed pharmacologically native-like [H-3]kainate binding properties. Further replacement of the membrane-embedded segments M1-M3 by a hydrophilic linker peptide gave rise to a soluble polypeptide which was accumulated in the culture medium. When bound to chelating Sepharose beads via a C-terminal histidine tag, the soluble fragment showed low-affinity binding of [H-3]kainate, which was displaced in a concentration-dependent manner by unlabelled domoic acid, L-glutamate and 6-cyano-7-nitroquinoxaline-2,3-dione. Our results indicate that the kainate-binding site is formed exclusively by the two discontinuous extracellular segments (S1 and S2) which are homologous to bacterial amino-acid-binding proteins. Ligand binding characteristics of soluble S1-S2 chimaeras between the GluR-6 and GluR-D subunits showed that, whereas both S1 and S2 segments contribute to agonist-selectivity, the N-terminal one-third of the GluR-D S2 segment is sufficient to confer alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-binding capacity to the chimaeric ligand-binding domain.