Cys-140 Is Critical for Metabotropic Glutamate Receptor-1 Dimerization*
Cys-140 Is Critical for Metabotropic Glutamate Receptor-1 Dimerization*
复制标题
DOI：
10.1074/jbc.m005581200
复制
发表时间：
2000-11
期刊：
影响因子：
--
通讯作者：
Kausik Ray;B. Hauschild
中科院分区：
文献类型：
--
作者：
Kausik Ray;B. Hauschild
代谢型谷氨酸受体 1 (mGluR1) 在细胞表面表达为二硫键连接的二聚体，可以用巯基试剂还原为单体。为了识别二聚化结构域，我们在 HEK-293 细胞中瞬时表达缺乏胞外结构域 (ECD) 的 mGluR1 (RhodC-R1) 的截短版本。在存在或不存在还原剂的情况下，RhodC-R1 都是单体，表明二聚化是通过 ECD 发生的。为了鉴定参与 ECD 内二聚化的半胱氨酸残基，在 ECD 氨基末端一半内的三个半胱氨酸处进行半胱氨酸到丝氨酸的点突变。 Cys-67、Cys-109 和 Cys-140 位点的突变在没有还原剂的情况下都会产生大量单体。单体C67S和C109S突变体没有正确糖基化，未能到达细胞表面，并且没有表现出谷氨酸反应，表明这些突变体受体折叠和/或加工不正确，因此保留在细胞内。相比之下，单体 C140S 突变体在细胞表面被正确糖基化、加工和表达。磷酸肌醇水解测定表明C140S突变型受体的谷氨酸反应与野生型受体相似。在 C140S 突变体背景上用半胱氨酸取代 Ser-129、Lys-134、Asp-143 和 Thr-146 可恢复受体二聚化。综上所述，结果表明 Cys-140 有助于 mGluR1 分子间二硫键连接的二聚化。
Metabotropic glutamate receptor 1 (mGluR1) expresses at the cell surface as disulfide-linked dimers and can be reduced to monomers with sulfhydryl reagents. To identify the dimerization domain, we transiently expressed in HEK-293 cells a truncated version of mGluR1 (RhodC-R1) devoid of the extracellular domain (ECD). RhodC-R1 was a monomer in the absence or presence of the reducing agents, suggesting that dimerization occurs via the ECD. To identify cysteine residues involved in dimerization within the ECD, cysteine to serine point mutations were made at three cysteines within the amino-terminal half of the ECD. A mutation at positions Cys-67, Cys-109, and Cys-140 all resulted in significant amounts of monomers in the absence of reducing agents. The monomeric C67S and C109S mutants were not properly glycosylated, failed to reach the cell surface, and showed no glutamate response, indicating that these mutant receptors were improperly folded and/or processed and thus retained intracellularly. In contrast, the monomeric C140S mutant was properly glycosylated, processed, and expressed at the cell surface. Phosphoinositide hydrolysis assay showed that the glutamate response of the C140S mutant receptor was similar to the wild type receptor. Substitution of a cysteine for Ser-129, Lys-134, Asp-143, and Thr-146 on the C140S mutant background restored receptor dimerization. Taken together, the results suggest that Cys-140 contributes to intermolecular disulfide-linked dimerization of mGluR1.