Fu D, Ballesteros J A, Weinstein H, Chen J, Javitch J A
Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Biochemistry. 1996 Sep 3;35(35):11278-85. doi: 10.1021/bi960928x.
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 that form the surface of the binding-site crevice in the third and fifth membrane-spanning segments (M3 and M5). We have now mutated to cysteine, one at a time, 26 consecutive residues in and flanking the seventh membrane-spanning segment (M7) and expressed the mutant receptors in HEK 293 cells. Nine of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist, sulpiride. Thus, we infer that the side chains of these residues are in the water-accessible surface of the binding-site crevice. The pattern of accessibility of the cysteine-substitution mutants is consistent with M7 being a kinked alpha-helix.
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，包含在其七个跨膜片段之间形成的一个可被水接触的裂隙内。我们之前利用半胱氨酸取代可及性方法，绘制了在第三和第五跨膜片段（M3和M5）中形成结合位点裂隙表面的残基图谱。我们现在已将第七跨膜片段（M7）及其侧翼的26个连续残基逐个突变为半胱氨酸，并在HEK 293细胞中表达了突变受体。这些突变体中有九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，并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，我们推断这些残基的侧链位于结合位点裂隙的可被水接触的表面。半胱氨酸取代突变体的可及性模式与M7是一个扭结的α螺旋一致。
