Purification and biochemical characterization of the D6 chemokine receptor
Purification and biochemical characterization of the D6 chemokine receptor
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DOI：
10.1042/bj20031266
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发表时间：
2004-04-15
影响因子：
4.1
通讯作者：
Graham, GJ
中科院分区：
文献类型：
--
作者：
Blackburn, PE;Simpson, CV;Graham, GJ
人们对趋化因子受体作为艾滋病、自身免疫和炎症性疾病以及癌症等疾病的治疗靶点非常感兴趣。阻碍此类研究的是这些分子缺乏准确的三维结构模型。 CC-趋化因子受体 D6 在异源转染子中以异常高的水平表达。在这里，我们报告了从相对较小的转染哺乳动物细胞培养物中毫克量的 D6 蛋白的纯化和生化表征。重要的是，纯化的 D6 保留了完整的功能活性，这通过 I-125 标记的 MIP-1ss（巨噬细胞炎症蛋白-1ss）的可置换结合以及受体与 MIP-1α 亲和柱的完全结合来证明。此外，我们还发现 D6 通过 N 连接糖基化修饰在 N 末端。突变分析表明，这种糖基化对于配体结合和转染细胞中的高表达来说是可有可无的。代谢标记显示受体也被硫酸化和磷酸化。磷酸化是配体独立的，并且不会通过配体结合和内化而增强，这表明与病毒趋化因子受体同源物 US28 相似。与 US28 一样，对转染细胞中 D6 的完整细胞补体的分析表明，> 80% 被发现与细胞内囊泡结构相关。这可能是这些细胞中可以合成大量 D6 的原因。 D6 的这些不寻常的特性以及此处描述的生化特征生成，引领了针对这种七次跨膜受体的三维结构的研究。
There is much interest in chemokine receptors as therapeutic targets in diseases such as AIDS, autoimmune and inflammatory disorders, and cancer. Hampering such studies is the lack of accurate three-dimensional structural models of these molecules. The CC-chemokine receptor D6 is expressed at exceptionally high levels in heterologous transfectants. Here we report the purification and biochemical characterization of milligram quantities of D6 protein from relatively small cultures of transfected mammalian cells. Importantly, purified D6 retains full functional activity, shown by displaceable binding of I-125-labelled MIP-1ss (macrophage inflammatory protein-1ss) and by complete binding of the receptor to a MIP-1alpha affinity column. In addition, we show that D6 is decorated on the N-terminus by N-linked glycosylation. Mutational analysis reveals that this glycosylation is dispensable for ligand binding and high expression in transfected cells. Metabolic labelling has revealed the receptor to also be sulphated and phosphorylated. Phosphorylation is ligand independent and is not enhanced by ligand binding and internalization, suggesting similarities with the viral chemokine receptor homologue US28. Like US28, an analysis of the full cellular complement of D6 in transfected cells indicates that > 80 % is found associated with intracellular vesicular structures. This may account for the high quantities of D6 that can be synthesized in these cells. These unusual properties of D6, and the biochemical characterization generating described here, leads the way towards work aimed at g the three-dimensional structure of this seven-transmembrane-spanning receptor.