Bai Ying, Lu Huafei, Machida Curtis A
Department of Integrative Biosciences, School of Dentistry, Oregon Health & Science University, Portland, OR 97239, USA.
Biochim Biophys Acta. 2006 Oct;1763(10):1076-89. doi: 10.1016/j.bbamcr.2006.08.009. Epub 2006 Aug 22.
The beta1-adrenergic receptor (beta1-AR) mRNAs are post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of its 3' untranslated region (UTR) with RNA binding proteins, including the HuR nuclear protein. In a previous report [Kirigiti et al. (2001). Mol. Pharmacol. 60:1308-1324], we examined the agonist-mediated down-regulation of the rat beta1-AR mRNAs, endogenously expressed in the rat C6 cell line and ectopically expressed in transfectant hamster DDT1MF2 and rat L6 cells. In this report, we determined that isoproterenol treatment of neonatal rat cortical neurons, an important cell type expressing beta1-ARs in the brain, results in significant decreases in beta1-AR mRNA stability, while treatment with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in significant increases in beta1-AR mRNA stability and nuclear retention. UV-crosslinking/immunoprecipitation and glycerol gradient fractionation analyses indicate that the beta1-AR 3' UTR recognize complexes composed of HuR and multiple proteins, including CRM 1. Cell-permeable peptides containing the leucine-rich nuclear export signal (NES) were used as inhibitors of CRM 1-mediated nuclear export. When DDT1MF2 transfectants were treated with isoproterenol and peptide inhibitors, only the co-addition of the NES inhibitor reversed the isoproterenol-induced reduction of beta1-AR mRNA levels. Our results suggest that CRM 1-dependent NES-mediated mechanisms influence the degradation and agonist-mediated down-regulation of the beta1-AR mRNAs.
β1 - 肾上腺素能受体（β1 - AR）mRNA在mRNA稳定性水平上受到转录后调控，并通过其3'非翻译区（UTR）与包括HuR核蛋白在内的RNA结合蛋白相互作用，经历激动剂介导的加速降解。在之前的一份报告[Kirigiti等人（2001年）。《分子药理学》60:1308 - 1324]中，我们研究了激动剂介导的大鼠β1 - AR mRNA的下调，该mRNA在大鼠C6细胞系中内源性表达，并在转染的仓鼠DDT1MF2和大鼠L6细胞中异位表达。在本报告中，我们确定，用异丙肾上腺素处理新生大鼠皮质神经元（大脑中表达β1 - AR的一种重要细胞类型）会导致β1 - AR mRNA稳定性显著降低，而用核输出受体CRM 1的抑制剂雷帕霉素B处理则会导致β1 - AR mRNA稳定性显著增加并在细胞核中滞留。紫外线交联/免疫沉淀和甘油梯度分级分析表明，β1 - AR 3' UTR识别由HuR和多种蛋白质（包括CRM 1）组成的复合物。含有富含亮氨酸的核输出信号（NES）的细胞可渗透肽被用作CRM 1介导的核输出的抑制剂。当用异丙肾上腺素和肽抑制剂处理DDT1MF2转染细胞时，只有同时添加NES抑制剂才能逆转异丙肾上腺素诱导的β1 - AR mRNA水平的降低。我们的结果表明，依赖CRM 1的NES介导的机制影响β1 - AR mRNA的降解和激动剂介导的下调。
