Williamson R A, Strange P G
Biological Laboratory, The University, Canterbury, Kent, England.
J Neurochem. 1990 Oct;55(4):1357-65. doi: 10.1111/j.1471-4159.1990.tb03147.x.
A series of group specific modifying reagents were tested for their effects on [3H]spiperone binding to brain D2 dopamine receptors to identify amino acid residues at the binding site of the D2 dopamine receptor that are critical for ligand binding. The dependence of ligand binding to the receptor on the pH of the incubation medium was also examined. N-Acetylimidazole, 5,5'-dithiobis(2-nitrobenzoic acid), 1,2-cyclohexanedione, and acetic anhydride had no specific effect on [3H]spiperone binding, indicating the lack of participation of tyrosine, free sulphydryl, arginine, or primary amino groups in ligand binding to the receptor. N,N'-Dicyclohexylcarbodiimide (DCCD) potently reduced the number of [3H]spiperone binding sites, indicating that a carboxyl group is involved in ligand binding to the receptor. The effects of DCCD could be prevented by prior incubation of the receptor with D2 dopamine receptor selective compounds. The pH-binding profile for [3H]spiperone binding indicated the importance of an ionising group of pKa 5.2 for ligand binding which may be the same carboxyl group. Diethyl pyrocarbonate, the histidine modifying reagent, also inhibited [3H]spiperone binding, reducing the affinity of the receptor for this ligand but the effects were not at the ligand binding site. From the effects of pH changes on ligand binding some evidence was obtained for a second ionising group (pKa 7.0) that specifically affects the binding of substituted benzamide drugs to the receptor. It is concluded that the D2 dopamine receptor binding site contains separate but over-lapping binding regions for antagonists such as spiperone and substituted benzamide drugs. The former region contains an important carboxyl group; the latter region contains another group that may be a second carboxyl group or a histidine.
测试了一系列组特异性修饰试剂对[³H]司来吉兰与脑D2多巴胺受体结合的影响，以确定D2多巴胺受体结合位点上对配体结合至关重要的氨基酸残基。还研究了配体与受体结合对孵育介质pH值的依赖性。N-乙酰咪唑、5,5'-二硫代双(2-硝基苯甲酸)、1,2-环己二酮和乙酸酐对[³H]司来吉兰结合没有特异性影响，表明酪氨酸、游离巯基、精氨酸或伯氨基不参与配体与受体的结合。N,N'-二环己基碳二亚胺(DCCD)显著减少了[³H]司来吉兰结合位点的数量，表明羧基参与了配体与受体的结合。DCCD的作用可通过受体与D2多巴胺受体选择性化合物预先孵育来预防。[³H]司来吉兰结合的pH结合曲线表明，pKa为5.2的电离基团对配体结合很重要，这可能是同一个羧基。组氨酸修饰试剂焦碳酸二乙酯也抑制了[³H]司来吉兰的结合，降低了受体对该配体的亲和力，但作用不在配体结合位点。从pH变化对配体结合的影响中获得了一些证据，表明存在第二个电离基团(pKa 7.0)，它特异性地影响取代苯甲酰胺药物与受体的结合。得出的结论是，D2多巴胺受体结合位点包含针对拮抗剂如司来吉兰和取代苯甲酰胺药物的独立但重叠的结合区域。前一个区域包含一个重要的羧基；后一个区域包含另一个基团，可能是第二个羧基或组氨酸。
