Defagot M C, Malchiodi E L, Villar M J, Antonelli M C
Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Buenos Aires, Argentina.
Brain Res Mol Brain Res. 1997 Apr;45(1):1-12. doi: 10.1016/s0169-328x(96)00235-5.
The distribution of the dopaminergic D4 receptor in rat brain was studied employing site directed polyclonal antibodies. Antisera were raised in rabbits to two oligopeptides corresponding to amino acids 160-172 of the second extracellular loop (P1) and amino acids 260-273 of the third intracellular loop (P2) of the D4 receptor sequence. Affinity-purified antibodies (anti-P1 and anti-P2) specifically recognized two major bands of 42-45 and 95 kDa in Western blots of denatured preparations of various rat brain areas. Immunocyto-chemistry studies showed that D4 receptor is widely distributed in rat central nervous system (CNS) showing higher labelling in the hippocampus (CA1, CA2, CA3 and dentate gyrus) frontal cortex, entorhinal cortex, caudate putamen, nucleus accumbens, olfactory tubercle, cerebellum, supraoptic nucleus and sustancia nigra pars compacta. In addition, anti-P1 decreased the binding of the antagonist [3H]YM-09151-2 selective for D2, D3 and D4 receptors but did not modify the binding of [3H]raclopride an antagonist selective for D2 and D3, in striatal synaptosomes. Anti-P2 did not modify the binding of these ligands. These results confirm the selectivity of the antibodies towards the D4 receptor and suggest that the binding site for the antagonists might be located at or close to the second extracellular loop of the protein sequence. D4 receptor protein is mainly expressed in plasma membranes and in the peripheral cytoplasm of neurons and is more widely distributed than was originally proposed based on mRNA localization, since it is present both in limbic, diencephalic and motor areas of rat brain.
采用位点定向多克隆抗体研究了多巴胺能D4受体在大鼠脑中的分布。用兔制备了针对与D4受体序列的第二个细胞外环（P1）的第160 - 172位氨基酸和第三个细胞内环（P2）的第260 - 273位氨基酸对应的两种寡肽的抗血清。亲和纯化抗体（抗P1和抗P2）在各种大鼠脑区变性制剂的蛋白质免疫印迹中特异性识别出42 - 45 kDa和95 kDa的两条主要条带。免疫细胞化学研究表明，D4受体广泛分布于大鼠中枢神经系统（CNS），在海马体（CA1、CA2、CA3和齿状回）、额叶皮质、内嗅皮质、尾状壳核、伏隔核、嗅结节、小脑、视上核和黑质致密部有较高的标记。此外，抗P1降低了对D2、D3和D4受体有选择性的拮抗剂[3H]YM - 09151 - 2的结合，但不改变对D2和D3有选择性的拮抗剂[3H]雷氯必利在纹状体突触体中的结合。抗P2不改变这些配体的结合。这些结果证实了抗体对D4受体的选择性，并表明拮抗剂的结合位点可能位于蛋白质序列的第二个细胞外环或其附近。D4受体蛋白主要表达于神经元的质膜和外周细胞质中，其分布比基于mRNA定位最初提出的更广泛，因为它存在于大鼠脑的边缘、间脑和运动区域。
