Leysen J E, Gommeren W, Mertens J, Luyten W H, Pauwels P J, Ewert M, Seeburg P
Department of Biochemical Pharmacology, Janssen Research Foundation, Beerse, Belgium.
Psychopharmacology (Berl). 1993;110(1-2):27-36. doi: 10.1007/BF02246947.
We investigated the ligand binding properties in vitro of two splice variants of the cloned human dopamine D2 receptor (the 443 and 414 amino acids long forms called D2L and D2S, respectively), expressed in 293 human kidney cells, in comparison with those of the dopamine D2 receptors in rat striatum, nucleus accumbens and tuberculum olfactorium. The new radioligand, [125I]2'-iodospiperone, showed a similar high binding affinity (KD:0.056-0.122 nM) for cloned human D2S and D2L receptors and for the D2 receptors in the three rat brain areas. Binding affinities of 25 dopamine antagonists and of 10 dopamine agonists belonging to different chemical classes were measured. The IC50 values of the antagonists were virtually identical in the five preparations: spiperone was the most potent compound (pIC50 approximately 9.9), remoxipride the least potent one (pIC50 approximately 5.7). The agonists showed similar IC50 values for the cloned human D2S and D2L receptors but their affinity for rat brain D2 receptors was 2- to 5-fold higher. Dopamine showed shallow inhibition curves, the high affinity binding was 10-fold lower for the cloned human D2 receptors than for the rat brain D2 receptors. Addition of stable guanosine-5'-triphosphate (GTP) analogues shifted the D2 receptors in the rat brain tissues to the "low" affinity state, the low affinity binding of dopamine was equal to the affinity for the cloned human receptor. None of the dopamine antagonists or agonists could differentiate between the two splice forms of the cloned human D2 receptors or between the D2 receptors in rat striatal and mesolimbic tissues. The lower apparent affinity of some agonists and of dopamine in the absence of stable GTP analogues suggests a less appropriate receptor G-protein coupling for the cloned human D2 receptors expressed in the 293 human kidney cells. Unexpectedly, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) reduced the [125I]2'-iodospiperone binding to the D2 receptors by 20-35% in the rat brain tissues and the cloned human D2L receptor, and by 75% to the cloned human D2S receptor. The inhibition in the last case could be prevented partly by submicromolar concentrations of dopamine. The GTP-gamma-S effect is suggested to be due to reduction of disulphide bonds in the receptor. Recent molecular modelling studies indicated an important role of the disulphide bridge between Cys107 at the start of transmembrane domain three and Cys182 in the third extracellular loop, for the binding of dopamine to the D2 receptor.
我们研究了在293人肾细胞中表达的克隆人多巴胺D2受体的两种剪接变体（分别为443个和414个氨基酸长的形式，称为D2L和D2S）的体外配体结合特性，并与大鼠纹状体、伏隔核和嗅结节中的多巴胺D2受体进行了比较。新型放射性配体[125I]2'-碘螺哌隆对克隆的人D2S和D2L受体以及大鼠脑三个区域中的D2受体显示出相似的高结合亲和力（KD：0.056 - 0.122 nM）。测定了25种属于不同化学类别的多巴胺拮抗剂和10种多巴胺激动剂的结合亲和力。拮抗剂的IC50值在五种制剂中几乎相同：螺哌隆是最有效的化合物（pIC50约为9.9），瑞莫必利是最无效的化合物（pIC50约为5.7）。激动剂对克隆的人D2S和D2L受体显示出相似的IC50值，但它们对大鼠脑D2受体的亲和力高2至5倍。多巴胺显示出浅抑制曲线，克隆的人D2受体的高亲和力结合比大鼠脑D2受体低10倍。添加稳定的鸟苷-5'-三磷酸（GTP）类似物使大鼠脑组织中的D2受体转变为“低”亲和力状态，多巴胺的低亲和力结合等于对克隆的人受体的亲和力。没有一种多巴胺拮抗剂或激动剂能够区分克隆的人D2受体的两种剪接形式或大鼠纹状体和中脑边缘组织中的D2受体。在没有稳定GTP类似物的情况下，一些激动剂和多巴胺较低的表观亲和力表明在293人肾细胞中表达的克隆人D2受体的受体G蛋白偶联不太合适。出乎意料的是，鸟苷-5'-O-（3-硫代三磷酸）（GTP-γ-S）使大鼠脑组织和克隆的人D2L受体中[125I]2'-碘螺哌隆与D2受体的结合减少20 - 35%，而使克隆的人D2S受体减少75%。在后一种情况下，亚微摩尔浓度的多巴胺可部分阻止这种抑制。GTP-γ-S的作用被认为是由于受体中二硫键的减少。最近的分子模拟研究表明，跨膜结构域三开始处的Cys107与第三个细胞外环中的Cys182之间的二硫桥对于多巴胺与D2受体的结合具有重要作用。
