Xu Lu, Sterling Carol R, Tank A William
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Mol Pharmacol. 2009 Oct;76(4):872-83. doi: 10.1124/mol.109.057596. Epub 2009 Jul 20.
Tyrosine hydroxylase (TH) plays a critical role in maintaining the appropriate concentrations of catecholamine neurotransmitters in brain and periphery, particularly during long-term stress, long-term drug treatment, or neurodegenerative diseases. Its expression is controlled by both transcriptional and post-transcriptional mechanisms. In a previous report, we showed that treatment of rat midbrain slice explant cultures or mouse MN9D cells with cAMP analog or forskolin leads to induction of TH protein without concomitant induction of TH mRNA. We further showed that cAMP activates mechanisms that regulate TH mRNA translation via cis-acting sequences within its 3'-untranslated region (UTR). In the present report, we extend these studies to show that MN9D cytoplasmic proteins bind to the same TH mRNA 3'-UTR domain that is required for the cAMP response. RNase T1 mapping demonstrates binding of proteins to a 27-nucleotide polypyrimidine-rich sequence within this domain. A specific mutation within the polypyrimidine-rich sequence inhibits protein binding and cAMP-mediated translational activation. UV-cross-linking studies identify a approximately 44-kDa protein as a major TH mRNA 3'-UTR binding factor, and cAMP induces the 40- to 42-kDa poly(C)-binding protein-2 (PCBP2) in MN9D cells. We show that PCBP2 binds to the TH mRNA 3'-UTR domain that participates in the cAMP response. Overexpression of PCBP2 induces TH protein without concomitant induction of TH mRNA. These results support a model in which cAMP induces PCBP2, leading to increased interaction with its cognate polypyrimidine binding site in the TH mRNA 3'-UTR. This increased interaction presumably plays a role in the activation of TH mRNA translation by cAMP in dopaminergic neurons.
酪氨酸羟化酶（TH）在维持大脑和外周儿茶酚胺神经递质的适当浓度方面起着关键作用，尤其是在长期应激、长期药物治疗或神经退行性疾病期间。其表达受转录和转录后机制的控制。在之前的一份报告中，我们表明用cAMP类似物或福斯可林处理大鼠中脑切片外植体培养物或小鼠MN9D细胞会导致TH蛋白的诱导，而不会同时诱导TH mRNA。我们进一步表明，cAMP激活了通过其3'-非翻译区（UTR）内的顺式作用序列调节TH mRNA翻译的机制。在本报告中，我们扩展了这些研究，以表明MN9D细胞质蛋白与cAMP反应所需的相同TH mRNA 3'-UTR结构域结合。核糖核酸酶T1图谱显示蛋白质与该结构域内一个富含27个核苷酸的多嘧啶序列结合。富含多嘧啶序列内的一个特定突变会抑制蛋白质结合和cAMP介导的翻译激活。紫外线交联研究确定一种约44 kDa蛋白是主要的TH mRNA 3'-UTR结合因子，并且cAMP在MN9D细胞中诱导40至42 kDa的聚（C）结合蛋白-2（PCBP2）。我们表明PCBP2与参与cAMP反应的TH mRNA 3'-UTR结构域结合。PCBP2的过表达诱导TH蛋白而不会同时诱导TH mRNA。这些结果支持了一个模型，其中cAMP诱导PCBP2，导致其与TH mRNA 3'-UTR中其同源多嘧啶结合位点的相互作用增加。这种增加的相互作用可能在多巴胺能神经元中cAMP激活TH mRNA翻译中起作用。
