Javitch J A, Ballesteros J A, Weinstein H, Chen J
Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Biochemistry. 1998 Jan 27;37(4):998-1006. doi: 10.1021/bi972241y.
The binding site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted-cysteine accessibility method, we previously mapped the residues in the third, fifth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 22 consecutive residues in the sixth membrane-spanning segment (M6) and expressed the mutant receptors in HEK 293 cells. Ten of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and all but one were protected from reaction by a reversible dopamine antagonist, sulpiride. Thus, we infer that the side chains of the residues at the reactive loci (V378, F382, W386, P388, F389, F390, T392, H393, I394, and I397) are on the water-accessible surface of the binding-site crevice. The pattern of accessibility is consistent with an alpha-helical conformation with a wide angle of accessibility near the binding site itself and a narrower stripe continuing toward the cytoplasmic portion of the binding-site crevice. This pattern of accessibility is consistent with the presence of a proline kink which could bend the extracellular portion of M6 into the binding-site crevice where it would be more broadly accessible than the cytoplasmic portion of the membrane-spanning segment. Four highly conserved aromatic residues and a histidine are clustered together on the water-accessible surface of the binding-site crevice. They define an interconnected "aromatic cluster" that may be involved in ligand binding and receptor activation.
多巴胺D2受体的结合位点，与其他同源G蛋白偶联受体的结合位点一样，位于其七个跨膜区段之间形成的一个可被水接触的裂隙内。我们先前利用半胱氨酸替代可及性方法，确定了第三、第五和第七跨膜区段中对该结合位点裂隙表面有贡献的残基。我们现在一次将第六跨膜区段（M6）中的22个连续残基突变为半胱氨酸，并在HEK 293细胞中表达突变受体。这些突变体中有10个与细胞外添加的带电荷、亲水性、疏脂性、巯基特异性试剂发生反应，除一个突变体外，其余所有突变体都受到可逆性多巴胺拮抗剂舒必利的保护而不发生反应。因此，我们推断反应位点（V378、F382、W386、P388、F389、F390、T392、H393、I394和I397）处残基的侧链位于结合位点裂隙的水可及表面。可及性模式与α-螺旋构象一致，在结合位点本身附近可及角较宽，而一条较窄的条带向结合位点裂隙的胞质部分延续。这种可及性模式与脯氨酸扭结的存在一致，脯氨酸扭结可将M6的细胞外部分弯曲到结合位点裂隙中，在那里它比跨膜区段的胞质部分更易于广泛接触。四个高度保守的芳香族残基和一个组氨酸聚集在结合位点裂隙的水可及表面。它们定义了一个相互连接的“芳香族簇”，可能参与配体结合和受体激活。
