Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity
Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity
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DOI：
10.1074/jbc.274.27.18902
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发表时间：
1999-07-02
影响因子：
4.8
通讯作者：
Parmentier, M
中科院分区：
文献类型：
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作者：
Blanpain, C;Lee, B;Parmentier, M
CCR5 是嗜巨噬细胞人类免疫缺陷病毒 I 型 (HIV-1) 的主要辅助受体。对于迄今为止已测试的大多数 G 蛋白偶联受体来说，连接细胞外环 (ECL) 的二硫键是维持配体结合和受体激活所需的结构完整性所必需的。影响 Cys(20) 的自然突变（被认为与 Cys(269) 形成二硫键）已在不同人群中得到描述，尽管这种突变对 CCR5 功能的影响尚不清楚。使用定点诱变，我们单独或组合突变了 CCR5 的四个胞外半胱氨酸，以研究它们在维持受体结构构象、其配体结合和信号转导特性及其作为病毒辅助受体的能力中的作用 60 。任何单个 Cys 残基的丙氨酸取代都会使表面表达水平降低 40-70%。然而，Cys(101) 或 Cys(178) 的突变预计会连接受体的 ECL1 和 ECL2，从而消除了一组构象敏感的抗 CCR5 抗体对 CCR5 的识别。突变对受体表达和构象的影响部分是温度敏感的，通过在 32°C 下孵育细胞实现了受体表达和构象的部分恢复。所有半胱氨酸突变体都无法 60 结合可检测水平的 MIP-1 β，并且对 CCR5 激动剂没有功能性反应。令人惊讶的是，所有半胱氨酸突变体确实支持 HIV R5 株的感染，尽管水平有所降低。这些结果表明，CCR5 的两个二硫键对于维持配体结合和信号传导所需的受体的结构完整性是必需的。 Env 结合和 HIV 进入机制似乎对 CCR5 构象的改变不太敏感。
CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation, A natural mutation affecting Cys(20), which is thought to form a disulfide bond with Cys(269), has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability 60 function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40-70%. However, mutation of Cys(101) or Cys(178), predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 degrees C. All cysteine mutants were unable 60 bind detectable levels of MIP-1 beta, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.