Choudhary M S, Craigo S, Roth B L
Department of Psychiatry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Mol Pharmacol. 1993 May;43(5):755-61.
The molecular processes by which agonists and antagonists bind to serotonin2 [5-hydroxytryptamine (5-HT2)] receptors are currently unknown. Three molecular models have proposed that conserved aromatic residues help to anchor the phenyl ring of 5-HT via stacking or pi-pi-type interactions with the 5-HT2 receptor. To test these models we made single point mutations (Phe339-->Leu339 and Phe340-->Leu340) of two aromatic residues that are conserved among all guanine nucleotide-binding protein-coupled 5-HT receptors and a single point mutation (Phe125-->Leu125) that exchanges a 5-HT2 for a 5-HT1c sequence. [3H]Mesulergine binding was abolished by Phe340-->Leu340 and unchanged with the Phe339-->Leu339 and Phe125-->Leu125 mutations, whereas [3H]ketanserin binding affinity was diminished by the Phe339-->Leu339 mutation and unchanged by Phe340-->Leu340 and Phe125-->Leu125. We also found that the affinities of three ergot derivatives (mesulergine, methysergide, and lisuride) were decreased by 88-1079-fold with only the Phe340-->Leu340 mutation. We also discovered that 4-[125I]iodo-2,5-(dimethoxy)phenylisopropylamine (DOI) binding was abolished in COS-7 cells expressing 5-HT2 (Phe340-->Leu340) receptors but maintained in cells expressing the Phe339-->Leu339 and Phe125-->Leu125 mutations. Additionally, the Ki values for several agonists and partial agonists (5-HT, DOI, m-chlorophenylpiperazine, trifluoromethylphenylpiperazine, bufotenine, and MK-212) were greatly diminished (26-14,000-fold decrease) only with the Phe340-->Leu340 receptor mutation. Finally, the Phe340-->Leu340 mutant receptors displayed an attenuated or abolished ability to augment phosphoinositide hydrolysis in COS-7 cells with four separate agonists (5-HT, MK-212, bufotenine, and quipazine). Taken together, these results are consistent with the idea that agonists and certain ergot derivatives anchor to 5-HT2 receptors, in part, via specific interactions with aromatic residue Phe340 located in transmembrane region VI.
目前尚不清楚激动剂和拮抗剂与5-羟色胺2 [5-羟色胺（5-HT2）]受体结合的分子过程。三种分子模型提出，保守的芳香族残基通过与5-HT2受体的堆积或π-π型相互作用，有助于锚定5-羟色胺的苯环。为了验证这些模型，我们对所有鸟嘌呤核苷酸结合蛋白偶联的5-羟色胺受体中保守的两个芳香族残基进行了单点突变（苯丙氨酸339→亮氨酸339和苯丙氨酸340→亮氨酸340），以及将5-HT2的一个单点突变（苯丙氨酸125→亮氨酸125）替换为5-HT1c序列。苯丙氨酸340→亮氨酸340使[3H]美舒麦角结合消失，而苯丙氨酸339→亮氨酸339和苯丙氨酸125→亮氨酸125突变对其无影响；而苯丙氨酸339→亮氨酸339突变使[3H]酮色林结合亲和力降低，苯丙氨酸340→亮氨酸340和苯丙氨酸125→亮氨酸125对其无影响。我们还发现，仅苯丙氨酸340→亮氨酸340突变就使三种麦角衍生物（美舒麦角、甲基麦角新碱和利苏瑞）的亲和力降低了88 - 1079倍。我们还发现，在表达5-HT2（苯丙氨酸340→亮氨酸340）受体的COS-7细胞中，4-[125I]碘-2,5-（二甲氧基）苯异丙胺（DOI）结合消失，但在表达苯丙氨酸339→亮氨酸339和苯丙氨酸125→亮氨酸125突变的细胞中仍保持。此外，仅苯丙氨酸340→亮氨酸340受体突变就使几种激动剂和部分激动剂（5-羟色胺、DOI、间氯苯哌嗪、三氟甲基苯哌嗪、蟾蜍色胺和MK-212）的Ki值大幅降低（降低26 - 14000倍）。最后，苯丙氨酸340→亮氨酸340突变型受体与四种不同激动剂（5-羟色胺、MK-212、蟾蜍色胺和喹哌嗪）一起在COS-7细胞中增强磷酸肌醇水解的能力减弱或消失。综上所述，这些结果与以下观点一致：激动剂和某些麦角衍生物部分通过与位于跨膜区VI的芳香族残基苯丙氨酸340的特异性相互作用，锚定在5-HT2受体上。
