Role of an AP-2-like element in transcriptional regulation of mouse μ-opioid receptor gene
Role of an AP-2-like element in transcriptional regulation of mouse μ-opioid receptor gene
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DOI：
10.1016/s0169-328x(03)00086-x
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发表时间：
2003-04-10
期刊：
影响因子：
--
通讯作者：
Loh, HH
中科院分区：
文献类型：
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作者：
Ko, JL;Liu, HC;Loh, HH
在小鼠μ阿片受体（莫尔）基因启动子近端，有几个重要的顺式元件和反式因子发挥着重要的作用。在本研究中，我们定义了另一个功能元件，位于近端启动子的-450到-400 bp（翻译起始位点指定为+1）区域，这也是完整启动子活性所必需的。它被命名为morAP-2样元件，因为它的序列与共有AP-2元件同源。令人惊讶的是，电泳迁移率变动分析（EMSA）显示，Sp1和Sp3，而不是AP-2蛋白，特异性结合的morAP-2样元素。导致Sp结合丧失的morAP-2样元件的突变导致活性降低约35%，进一步证实了morAP-2样元件在莫尔基因表达中的积极作用。SP蛋白与碱性磷酸酶的去磷酸化也降低SP结合的morAP-2样元素在EMSA中，这表明磷酸化的SP是必不可少的，其结合到这个元素。然而，直接或间接激活PKA（一种经典的G-蛋白偶联信号通路）并不导致SP与morAP-2样元件结合的显著变化，也不导致表达莫尔的SH-SY 5 Y细胞的启动子活性的显著变化，这表明SP的磷酸化不涉及PKA。这些结果表明，结合不同的磷酸化形式的Sp蛋白的morAP-2样元件可能有助于微调的莫尔在不同的细胞中的表达。(C)2003 Elsevier Science B. V.保留所有权利。
Previously, several important cis-elements and trans-factors have been shown to play a functional role in the proximal promoter of mouse mu-opioid receptor (MOR) gene. In this study, we defined another functional element located the in -450 to -400 bp (translational start site designated as +l) region of the proximal promoter, which is also essential for the full promoter activity. It is designated as the morAP-2-like element for its sequence homologous to the consensus AP-2 element. Surprisingly, electrophoretic mobility shift analysis (EMSA) revealed that Sp1 and Sp3, but not AP-2 proteins, were specifically bound to the morAP-2-like element. Mutation of the morAP-2-like element, resulting in a loss of Sp binding, led to an approximately 35% decrease in activity, further confirming the positive role of the morAP-2-like element in MOR gene expression. Dephosphorylation of Sp proteins with alkaline phosphatase also decreased Sp binding to the morAP-2-like element in EMSA, suggesting phosphorylation of Sp is essential for its binding to this element. However, direct or indirect activation of PKA, a classical G-protein coupled signaling pathway, resulted in no significant change of Sp binding to the morAP-2-like element, nor of the promoter activity the SH-SY5Y cells, MOR expressing cells, suggesting that phosphorylation of Sp does not involve PKA. These results suggest that the binding of different phosphorylated forms of Sp proteins to the morAP-2-like element may contribute to the fine tuning of MOR expression in different cells. (C) 2003 Elsevier Science B.V. All rights reserved.