Plug M J, Dijk J, Maassen J A, Möller W
Department of Medical Biochemistry, Sylvius Laboratory University of Leiden, The Netherlands.
Eur J Biochem. 1992 May 15;206(1):123-30. doi: 10.1111/j.1432-1033.1992.tb16909.x.
The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.
使用D2受体特异性激动剂N-0434，通过麦胚凝集素-琼脂糖凝胶层析和亲和层析法纯化牛多巴胺D2受体。纯化得到一种主要蛋白条带为95 kDa的制剂。为了确定该蛋白的身份，基于克隆的D2受体的预测氨基酸序列，使用合成肽制备了针对多巴胺D2受体的多克隆抗体。为了初步筛选这些抗体，构建了三种由β-半乳糖苷酶和受体片段组成的融合蛋白。一种抗血清在蛋白质印迹和免疫沉淀实验中均与相应的D2受体融合蛋白发生强烈反应。在每种情况下，与游离肽的竞争均可抑制识别。在牛纹状体部分纯化的受体制剂的蛋白质印迹上，该抗血清特异性识别一种95 kDa的糖蛋白。在类似制剂中，该抗血清沉淀出相当一部分活性D2受体，这通过上清液中[3H]螺哌隆结合的减少来确定。通过添加游离肽可从免疫沉淀物中释放出活性受体。对瞬时转染了编码D2受体的DNA的细胞进行免疫细胞化学分析，结果显示转染细胞有特异性染色。针对第三细胞内环中的序列产生的抗体能够将受体对多巴胺的亲和力从高转变为低，这表明该抗血清可能干扰受体与GTP结合蛋白的相互作用。
