SITE-DIRECTED MUTAGENESIS OF THE HUMAN DOPAMINE-D2 RECEPTOR
SITE-DIRECTED MUTAGENESIS OF THE HUMAN DOPAMINE-D2 RECEPTOR
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DOI：
10.1016/0922-4106(92)90129-j
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发表时间：
1992-10-01
期刊：
影响因子：
--
通讯作者：
AKIL, H
中科院分区：
文献类型：
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作者：
MANSOUR, A;MENG, F;AKIL, H
基于氨基酸序列和计算机建模，多巴胺D2受体的两个相互冲突的三维模型已经提出。一个模型（Dahl等人，1991，Proc.Natl. Acad. Sci. USA 88，8111）表明多巴胺与跨膜（TM）2的天冬氨酸80和TM 6的天冬酰胺390相互作用，跨膜以顺时针方式排列，而第二个模型（Hibert等人，1991，Mol. Pharmacol. 40，8）表明多巴胺与TM 3的天冬氨酸114和TM 5的丝氨酸（194和197）相互作用，当从细胞外空间观察时，跨膜以逆时针方式排列。本研究测试后一种模型通过选择性突变天冬氨酸114和丝氨酸194和197的人多巴胺D2受体的定点诱变。此外，突变两个蛋氨酸（116和117），以评估多巴胺D2受体的天冬氨酸（114）附近的残基是否是区分多巴胺受体激动剂和肾上腺素受体激动剂的关键。随着天冬氨酸（114）突变为天冬酰胺或甘氨酸，去除负电荷导致激动剂和拮抗剂结合的完全丧失。在位置116和117的单个或双甲硫氨酸突变，使多巴胺D2结合口袋更接近β-2-肾上腺素受体，没有导致对去甲肾上腺素能激动剂或拮抗剂的选择性的变化。丝氨酸突变揭示了多巴胺D2受体和肾上腺素受体之间有趣的差异。特别是，丝氨酸197似乎比丝氨酸194更重要的激动剂结合。此外，一种激动剂（N-0437）的结合不受单个丝氨酸突变的影响，而一些拮抗剂（如雷氯必利和螺哌隆）的结合则显著改变。这些研究结果进行了讨论，配体结构和它们的相互作用与假定的结合口袋。
Based on amino acid sequence and computer modeling, two conflicting three-dimensional models of the dopamine D2 receptor have been proposed. One model (Dahl ct al., 1991, Proc. Natl. Acad. Sci. USA 88, 8111) suggests that dopamine interacts with aspartate 80 of transmembrane (TM) 2 and asparagine 390 of TM6 with the transmembranes arranged in a clockwise manner, while a second model (Hibert et al., 1991, Mol. Pharmacol. 40, 8) suggests that dopamine interacts with aspartate 114 of TM3 and the serines of TM5 (194 and 197) with the transmembranes arranged in a counterclockwise manner when viewed from the extracellular space. The present study tests the latter model by selectively mutating aspartate 114 and serines 194 and 197 of the human dopamine D2 receptor by site-directed mutagenesis. In addition, two methionines (116 and 117) were mutated to evaluate whether residues near aspartate (114) of the dopamine D2 receptor arc critical in differentiating dopamine receptor agonists from adrenoceptor agonists. Removal of the negative charge with the mutation of aspartate (114) to either asparagine or glycine led to a total loss of both agonist and antagonist binding. Individual or dual methionine mutations in positions 116 and 117, to make the dopamine D2 binding pocket more closely resemble the beta-2-adrenoccptor, did not result in a change in selectivity toward noradrenergic agonists or antagonists. The serine mutations revealed interesting differences between the dopamine D2 receptor and the adrenoceptors. In particular, serine 197 appeared more important than serine 194 for agonist binding. In addition, the binding of one agonist (N-0437) was unaffected by individual serine mutations, while the binding of some antagonists, such as raclopride and spiperone, was significantly altered. These findings are discussed in relation to ligand structure and their interactions with the putative binding pocket.