An aromatic microdomain at the cannabinoid CB1 receptor constitutes an agonist/inverse agonist binding region
An aromatic microdomain at the cannabinoid CB1 receptor constitutes an agonist/inverse agonist binding region
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DOI：
10.1021/jm0302647
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发表时间：
2003-11-20
影响因子：
7.3
通讯作者：
Abood, ME
中科院分区：
文献类型：
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作者：
McAllister, SD;Rizvi, G;Abood, ME
大麻素CB受体跨膜螺旋（TMH）3-4-5-6区包括由残基F3.25、F3.36、W4.64、Y5.39、W5.43和W6.48组成的芳香族微结构域。在以前的工作中，我们已经证明了CB中5.39位的芳香性对于CB 1的正常功能至关重要。本文报道的建模研究表明，在CB 1的非活性状态下，CB 1反向激动剂/拮抗剂SR 141716 A的结合位点位于TMH 3 -4-5-6芳香族微结构域内，并涉及与F3.36、Y5.39和W5.43的直接芳香族堆积相互作用以及与K3.28的氢键结合。此外，建模研究表明，在CB 1的活性状态下，CB激动剂WIN 55，212-2在相同的芳香族微结构域中结合，与F3.36，W5.43和W6.48直接芳香族堆积相互作用。相比之下，在结合口袋模型中，CB激动剂大麻素结合在TMH 2 -3-6-7区域，其中氢键和C-H（...）π相互作用似乎是重要的。只有一个TMH 3芳香族残基，F3.25，被发现是花生四烯酸结合口袋的一部分。为了探测TMH 3 - 4 - 5 - 6芳香族微结构域对配体结合的重要性，产生稳定转染的细胞系，用于将每个芳香族微结构域残基单点突变为丙氨酸。观察到W4.64A的细胞表达不当，并排除了对该突变的进一步表征。大麻素激动剂CP 55，940的亲和力不受F3.25A、F3.36A、W5.43A或W6.48A突变的影响，使得CP 55，940成为结合研究的放射性配体的适当选择。发现SR 141716 A和WIN 55，212-2的结合受到F3.36A、W5.43A和W6.48A突变的影响，表明这些残基是这两种配体结合位点的一部分。仅发现F3.25A突变影响大麻素的结合，表明大麻素的结合位点区域与WIN 55，212-2以及SR 141716 A不同。总之，这些结果支持将TMH 3 -4-5-6芳香族微结构域鉴定为SR 141716 A和WIN 55，212-2的结合区域而不是大麻素的结合区域的建模研究。
The cannabinoid CB, receptor transmembrane helix (TMH) 3-4-5-6 region includes an aromatic microdomain comprised of residues F3.25, F3.36, W4.64, Y5.39, W5.43, and W6.48. In previous work, we have demonstrated that aromaticity at position 5.39 in CB, is crucial for proper function of CB1. Modeling studies reported here suggest that in the inactive state of CB1, the binding site of the CB1 inverse agonist/antagonist SR141716A is within the TMH3-4-5-6 aromatic microdomain and involves direct aromatic stacking interactions with F3.36, Y5.39, and W5.43, as well as hydrogen bonding with K3.28. Further, modeling studies suggest that in the active state of CB1, the CB agonist WIN55,212-2 binds in this same aromatic microdomain, with direct aromatic stacking interactions with F3.36, W5.43, and W6.48. In contrast, in the binding pocket model, the CB agonist anandamide binds in the TMH2-3-6-7 region in which hydrogen bonding and C-H(...)pi interactions appear to be important. Only one TMH3 aromatic residue, F3.25, was found to be part of the anandamide binding pocket. To probe the importance of the TMH3 - 4 - 5 - 6 aromatic microdomain to ligand binding, stable transfected cell lines were created for single-point mutations of each aromatic microdomain residue to alanine. Improper cellular expression of the W4.64A was observed and precluded further characterization of this mutation. The affinity of the cannabinoid agonist CP55,940 was unaffected by the F3.25A, F3.36A, W5.43A, or W6.48A mutations, making CP55,940 an appropriate choice as the radioligand for binding studies. The binding of SR141716A and WIN55,212-2 were found to be affected by the F3.36A, W5.43A, and W6.48A mutations, suggesting that these residues are part of the binding site for these two ligands. Only the F3.25A mutation was found to affect the binding of anandamide, suggesting a divergence in binding site regions for anandamide from WIN55,212-2, as well as SR141716A. Taken together, these results support modeling studies that identify the TMH3-4-5-6 aromatic microdomain as the binding region of SR141716A and WIN55,212-2, but not of anandamide.