Potenza M N, Graminski G F, Schmauss C, Lerner M R
Department of Cell Biology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
J Neurosci. 1994 Mar;14(3 Pt 2):1463-76. doi: 10.1523/JNEUROSCI.14-03-01463.1994.
Functional characteristics of human D2 and D3 receptors (DRs) were examined using a new bioassay suited for the study of Gi-protein-coupled receptors (GiRs). The bioassay utilizes pigment granule aggregation within cultured Xenopus laevis melanophores for the quantitative evaluation of ligands as agonists or antagonists upon particular GiRs. Initial feasibility studies were performed by analyzing a melanocyte receptor endogenous to the melanophores. In dose-dependent manners, melatonin inhibited melatonin-stimulating hormone-induced cAMP accumulation and caused pigment aggregation that could be monitored over time. Next, melanophores were transiently transfected with cDNAs coding for the human D2BR (short form) and D3R. Expression of either receptor conferred upon the cells the ability to aggregate their melanosomes in response to selective dopaminergic agonists. The same ligands also inhibited cAMP accumulation within the transfected melanophores, and the agonist-induced pigment aggregation was shown to be sensitive to pertussis toxin. EC50 and IC50 value determinations revealed that agonists activated the D2R and D3R at similar concentrations, while each of the antagonists displaying an effect was more potent upon the D2R. The results reveal functional similarities and differences between the D2R and D3R.
利用一种适用于研究Gi蛋白偶联受体（GiRs）的新生物测定法，检测了人类D2和D3受体（DRs）的功能特性。该生物测定法利用培养的非洲爪蟾黑素细胞内的色素颗粒聚集，对特定GiRs的配体作为激动剂或拮抗剂进行定量评估。通过分析黑素细胞内源性的黑素细胞受体进行了初步可行性研究。褪黑素以剂量依赖的方式抑制褪黑素刺激激素诱导的cAMP积累，并导致色素聚集，这种聚集可随时间进行监测。接下来，用编码人类D2BR（短形式）和D3R的cDNA对黑素细胞进行瞬时转染。任一受体的表达赋予细胞响应选择性多巴胺能激动剂而聚集其黑素小体的能力。相同的配体也抑制转染黑素细胞内的cAMP积累，并且激动剂诱导的色素聚集对百日咳毒素敏感。EC50和IC50值的测定表明，激动剂在相似浓度下激活D2R和D3R，而显示出作用的每种拮抗剂对D2R的作用更强。结果揭示了D2R和D3R之间的功能异同。
