Cysteine residues in the human cannabinoid receptor: Only C257 and C264 are required for a functional receptor, and steric bulk at C386 impairs antagonist SR141716A binding
Cysteine residues in the human cannabinoid receptor: Only C257 and C264 are required for a functional receptor, and steric bulk at C386 impairs antagonist SR141716A binding
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DOI：
10.1021/bi0472651
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发表时间：
2005-06-21
期刊：
影响因子：
2.9
通讯作者：
Farrens, DL
中科院分区：
文献类型：
--
作者：
Fay, JF;Dunham, TD;Farrens, DL
人类神经元大麻素受体(CBI)是一种G蛋白偶联受体(GPCR)，由大麻中的精神活性成分以及大脑中产生的内源性大麻素触发。与大多数GPCR一样，CB1激活的机制尚不清楚。在这项工作中，我们评估了半胱氨酸残基在CB1配体结合和激活中的作用，并展示了一种定位CBI结构和功能中关键决定因素的方法。通过突变分析，我们发现只有两个半胱氨酸C257和C264是高水平表达和受体功能所需的。此外，通过半胱氨酸反应性研究，我们发现跨膜螺旋7上的半胱氨酸C386(C7.42)与甲硫磺酸盐(MTS)巯基标记剂反应，因此是溶剂可及的。有趣的是，通过NITS标记或突变在该位置引入的立体块抑制了拮抗剂药物SR141716A(也称为利莫那班或AcComplia)的结合，但不影响激动剂CP55940的结合。我们随后的建模研究表明，这种影响是由于修饰的C386残基与SR141716A的哌啶环发生空间碰撞和/或结合口袋中的芳香微域被破坏所致。在这些结果的基础上，我们假设结合SR141716A抑制了跨膜螺旋6在功能活跃的受体状态形成期间的移动能力。
The human neuronal cannabinoid receptor (CBI) is a G-protein-coupled receptor (GPCR) triggered by the psychoactive ingredients in marijuana, as well as endogenous cannabinoids produced in the brain. As with most GPCRs, the mechanism of CB1 activation is poorly understood. In this work, we have assessed the role of cysteine residues in CB1 ligand binding and activation, and demonstrate a method for mapping key determinants in CBI structure and function. Through mutational analysis, we find that only two cysteines, C257 and C264, are required for high-level expression and receptor function. In addition, through cysteine reactivity studies, we find that a cysteine in transmembrane helix seven, C386 (C7.42), is reactive toward methanethiosulfonate (MTS) sulfhydryl labeling agents, and is thus solvent accessible. Interestingly, steric bulk introduced at this site, either through NITS labeling or by mutation, inhibits binding of the antagonist drug SR141716A (also known as Rimonabant or Accomplia), but does not affect the binding of the agonist CP55940. Our subsequent modeling studies suggest this effect is caused by steric clash of the modified C386 residue with the piperidine ring of SR141716A and/or disruption of an aromatic microdomain in the binding pocket. On the basis of these results, we hypothesize that bound SR141716A inhibits the ability of transmembrane helix 6 to move during formation of the functionally active receptor state.