Minowa M T, Lee S H, Mouradian M M
Genetic Pharmacology Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
DNA Cell Biol. 1996 Sep;15(9):759-67. doi: 10.1089/dna.1996.15.759.
Stimulation of the D1A dopamine receptor increases intracellular cAMP concentration and down-regulates the receptor protein. We evaluated the possibility that this second messenger system could affect the expression of the D1A gene as a positive autoregulatory mechanism. Treatment of the D1A-expressing cells SK-N-MC with 100 microM dopamine resulted in an initial increase in steady-state levels of the D1A mRNA beginning at 30 min, followed by decline below the baseline and then recovery by 24 hr. Forskolin/IBMX (100 microM each) treatment also resulted in a decline followed by recovery. To determine if these changes in D1A message levels are due to transcriptional control, transient expression assays were done using reporter gene constructs of the human D1A gene 5'-flanking region. Forskolin/IBMX treatment for 19 hr resulted in a four- to seven-fold increase in trans-activation of the human D1A gene promoter. Two cAMP-responsive regions in exon 1 of this gene with nuclear protein binding sites within both regions were identified. The segment of the D1A gene between these two cAMP-responsive regions contained two additional DNA-protein interaction sites, one of which bound to nuclear factors considerably stronger following forskolin/IBMX treatment. Several consensus sequences for classical transcription factors known to mediate the cAMP response, such as CREB, AP2, and AP1, are found in the human D1A gene. However, the location of all but one AP2 site in other parts of this gene and lack of AP2 expression in SK-N-MC cells suggest that these factors are unlikely to transduce this response. Thus, dopamine treatment results in delayed cAMP-mediated trans-activation of the D1A gene via an indirect mechanism.
刺激 D1A 多巴胺受体可增加细胞内 cAMP 浓度并下调受体蛋白。我们评估了这种第二信使系统作为一种正向自调节机制影响 D1A 基因表达的可能性。用 100 μM 多巴胺处理表达 D1A 的细胞 SK-N-MC，导致 D1A mRNA 稳态水平在 30 分钟时开始最初升高，随后降至基线以下，然后在 24 小时时恢复。福斯高林/异丁基甲基黄嘌呤（各 100 μM）处理也导致先下降后恢复。为了确定 D1A 信使水平的这些变化是否归因于转录控制，使用人 D1A 基因 5' 侧翼区的报告基因构建体进行了瞬时表达分析。福斯高林/异丁基甲基黄嘌呤处理 19 小时导致人 D1A 基因启动子的反式激活增加了 4 至 7 倍。在该基因外显子 1 中鉴定出两个 cAMP 反应区域，两个区域内均有核蛋白结合位点。这两个 cAMP 反应区域之间的 D1A 基因片段包含另外两个 DNA - 蛋白质相互作用位点，其中一个在福斯高林/异丁基甲基黄嘌呤处理后与核因子的结合明显增强。在人 D1A 基因中发现了几种已知介导 cAMP 反应的经典转录因子的共有序列，如 CREB、AP2 和 AP1。然而，除了一个 AP2 位点外，其他所有 AP2 位点都位于该基因的其他部位，并且 SK-N-MC 细胞中缺乏 AP2 表达，这表明这些因子不太可能转导这种反应。因此，多巴胺处理通过间接机制导致 D1A 基因的 cAMP 介导的延迟反式激活。
