Dopamine D4/D2 receptor selectivity is determined by a divergent aromatic microdomain contained within the second, third, and seventh membrane-spanning segments
Dopamine D4/D2 receptor selectivity is determined by a divergent aromatic microdomain contained within the second, third, and seventh membrane-spanning segments
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DOI：
10.1124/mol.56.6.1116
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发表时间：
1999-12-01
影响因子：
3.6
通讯作者：
Javitch, JA
中科院分区：
文献类型：
--
作者：
Simpson, MM;Ballesteros, JA;Javitch, JA
多巴胺受体和同源G蛋白偶联受体序列的保守特征指向有助于其配体结合位点的区域以及这些区域内的氨基酸残基。然而，儿茶酚胺受体之间结合特异性的差异必定源于它们的非保守残基。使用取代半胱氨酸可及性方法，我们鉴定了形成多巴胺 D2 受体中可水结合位点缝隙表面的残基。在 D2 和 D4 受体之间大约 80 个不同的跨膜残基中，只有 20 个被发现是可接近的，并且这 20 个中的 6 个是保守的脂肪族取代。在 D2 受体背景中，我们将 14 个可接近的非保守残基单独或组合突变为 D4 受体中对齐的残基。我们还在 D4 受体背景中进行了相互突变。这些残基中的四到六个残基的组合取代足以将受体对几种化学上不同的D4选择性拮抗剂的亲和力在两个方向上改变三个数量级（D2-到D4-样和D4-到D2-样）。突变残基位于第二、第三和第七跨膜片段（M2、M3、M7）中，并在结合位点缝隙中形成簇。 M2 中该簇中单个残基的突变足以将氯氮平的亲和力增加至 D4 样水平。我们可以根据配体中与 D2 和 D4 受体 M2-M3-M7 中不同的芳香微结构域相互作用的一组化学部分来合理化数据。
Conserved features of the sequences of dopamine receptors and of homologous G-protein-coupled receptors point to regions, and amino acid residues within these regions, that contribute to their ligand binding sites. Differences in binding specificities among the catecholamine receptors, however, must stem from their nonconserved residues. Using the substituted-cysteine accessibility method, we have identified the residues that form the surface of the water-accessible binding-site crevice in the dopamine D2 receptor. Of approximately 80 membrane-spanning residues that differ between the D2 and D4 receptors, only 20 were found to be accessible, and 6 of these 20 are conservative aliphatic substitutions. In a D2 receptor background, we mutated the 14 accessible, nonconserved residues, individually or in combinations, to the aligned residues in the D4 receptor. We also made the reciprocal mutations in a D4 receptor background. The combined substitution of four to six of these residues was sufficient to switch the affinity of the receptors for several chemically distinct D4-selective antagonists by three orders of magnitude in both directions (D2- to D4-like and D4- to D2-like). The mutated residues are in the second, third, and seventh membrane-spanning segments (M2, M3, M7) and form a cluster in the binding-site crevice. Mutation of a single residue in this cluster in M2 was sufficient to increase the affinity for clozapine to D4- like levels. We can rationalize the data in terms of a set of chemical moieties in the ligands interacting with a divergent aromatic microdomain in M2-M3-M7 of the D2 and D4 receptors.