Plouffe Bianca, D'Aoust Jean-Philippe, Laquerre Vincent, Liang Binhui, Tiberi Mario
Ottawa Hospital Research Institute (Neurosciences), Department of Medicine, University of Ottawa, Ontario, Canada.
Methods Enzymol. 2010;484:295-328. doi: 10.1016/B978-0-12-381298-8.00016-2.
Dopamine D1 and D5 receptors are prototypical cell-surface seven-transmembrane (TM) G protein-coupled receptors (GPCRs) mediating elevation of intracellular cAMP levels. The high level of constitutive activity of D5 receptor mediating intracellular cAMP production is one of the functional hallmarks distinguishing the closely related D1-like dopaminergic subtypes (D1 and D5). D1-like subtypes share over 80% identity within their TM regions. Thus, D1 and D5 receptors can serve as unparalleled and useful molecular tools to gain structural and mechanistic insights into subtype-specific determinants regulating GPCR constitutive activation and inverse agonism. A method has been developed that relies on the use of transfected human embryonic kidney 293 cells with wild-type (WT), epitope-tagged, chimeric, truncated, and mutant forms of mammalian D1 and D5 receptors using a modified DNA and calcium phosphate precipitation procedure. Receptor expression levels are quantified by a radioligand binding using [(3)H]-SCH23390, a D1-like selective drug. Regulation of ligand-independent and dependent activity of WT and mutated D1 and D5 receptors is determined by whole cell cAMP assays using metabolic [(3)H]-adenine labeling and sequential purification radiolabeled nucleotides over Dowex and alumina resin columns. Results on the regulation of D1 and D5 constitutive activity are presented here. Our studies indicate that dopamine-mediated D5 receptor stimulation in a dose-dependent manner is not always detectable, suggesting that D5 receptors can exist in a "locked" constitutively activated state. This "locked" constitutively active state of D5 receptor is not linked to aberrant high receptor expression levels or cell behavior, as D1 receptor function remains essentially unchanged in these cells. In fact, we show that phorbol ester treatment of cells harboring "locked" constitutively active D5 receptors abrogates constitutive activation of D5R to allow its stimulation by dopamine in a dose-dependent manner.
多巴胺 D1 和 D5 受体是典型的细胞表面七跨膜 (TM) G 蛋白偶联受体 (GPCR)，可介导细胞内 cAMP 水平升高。D5 受体介导细胞内 cAMP 产生的高水平组成性活性是区分密切相关的 D1 样多巴胺能亚型（D1 和 D5）的功能特征之一。D1 样亚型在其跨膜区域内具有超过 80% 的同一性。因此，D1 和 D5 受体可作为无与伦比且有用的分子工具，以深入了解调节 GPCR 组成性激活和反向激动作用的亚型特异性决定因素的结构和机制。已开发出一种方法，该方法依赖于使用经修饰的 DNA 和磷酸钙沉淀程序，转染具有野生型 (WT)、表位标记、嵌合、截短和突变形式的哺乳动物 D1 和 D5 受体的人胚肾 293 细胞。使用 D1 样选择性药物 [(3)H]-SCH23390 通过放射性配体结合对受体表达水平进行定量。使用代谢 [(3)H]-腺嘌呤标记并通过 Dowex 和氧化铝树脂柱对放射性标记核苷酸进行连续纯化，通过全细胞 cAMP 测定法确定 WT 和突变的 D1 和 D5 受体的配体非依赖性和依赖性活性的调节。本文展示了关于 D1 和 D5 组成性活性调节的结果。我们的研究表明，多巴胺介导的 D5 受体刺激并不总是呈剂量依赖性可检测到，这表明 D
