Intragenic sequences of the human glucocorticoid receptor complementary DNA mediate hormone-inducible receptor messenger RNA down-regulation through multiple mechanisms.
Intragenic sequences of the human glucocorticoid receptor complementary DNA mediate hormone-inducible receptor messenger RNA down-regulation through multiple mechanisms.
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DOI：
10.1210/mend.8.12.7708063
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发表时间：
1994-12
影响因子：
--
通讯作者：
K. Burnstein;C. Jewell;M. Sar;J. Cidlowski
中科院分区：
文献类型：
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作者：
K. Burnstein;C. Jewell;M. Sar;J. Cidlowski
糖皮质激素受体(GR)是一类配体依赖的转录因子，在细胞生长、分化和死亡的内分泌调控中发挥重要作用。人们普遍认为，这些类固醇受体在细胞培养和动物(包括人类)中接触配体后会发生下调。这种细胞受体水平的降低导致对随后的激素注射不敏感。然而，控制GR同源下调的机制却知之甚少。我们先前已经证明：(1)转染人GR(HGR)互补DNA(CDNA)包含的序列足以概括体内HGR信使RNA(MRNA)和蛋白质的下调。我们现在已经评估了HGR mRNA的激素调节的潜在机制，并进一步确定了HGR基因的一个包含下调信号的基因内域。在放线菌素-D或放线菌素-D存在的情况下，糖皮质激素处理表达hGR基因的COS-1细胞可下调hGR基因的表达，提示糖皮质激素诱导蛋白不是下调所必需的。我们发现，受体被配体长时间占据会导致GR mRNA转换率增加，此外，激动剂地塞米松或拮抗剂RU486都会降低HGR基因的转录。为了确定哪些受体序列在下调中起关键作用，采用了一系列hGR基因缺失突变体与全长hGR基因一起共转染的策略。检测糖皮质激素对突变受体cDNAs编码的受体mRNAs的调节作用。受体cDNA5‘端缺失产生的转录本容易受到糖皮质激素的下调，而位于受体编码序列3’端的序列(对应于550-697氨基酸)的缺失导致受体转录本仅受糖皮质激素最小程度的下调。总之，这些研究表明，多种机制控制着GR mRNA的丰度，而配体结合域中的基因内元件是这种下调的关键。
Glucocorticoid receptors (GR) are ligand-dependent transcription factors that play a critical role in the endocrine control of cell growth, differentiation, and death. These steroid receptors are widely recognized to undergo down-regulation after exposure to ligand in cell cultures and animals, including humans. This reduction in cellular receptor levels leads to insensitivity to subsequent hormone administration. The mechanisms controlling homologous down-regulation of the GR are, however, poorly understood. We have previously shown (1) that a transfected human GR (hGR) complementary DNA (cDNA) contains sequences that are sufficient to recapitulate the down-regulation of both hGR messenger RNA (mRNA) and protein seen in vivo. We have now evaluated potential mechanisms involved in the hormonal regulation of the hGR mRNA and, further, have identified an intragenic domain of the hGR cDNA that contains the down-regulatory signal. Glucocorticoid treatment of COS-1 cells expressing a transfected hGR cDNA resulted in down-regulation of the hGR mRNA in the presence of cycloheximide or actinomycin-D, suggesting that a glucocorticoid-inducible protein was not essential for down-regulation. We show that prolonged receptor occupation by ligand leads to increased GR mRNA turnover, and furthermore, that either the agonist dexamethasone or the antagonist RU486 decreased transcription of the hGR cDNA. To resolve which receptor cDNA sequences are critical in down-regulation, a cotransfection strategy was employed in which a series of hGR cDNA deletion mutants was transfected in conjunction with the full-length hGR cDNA. The effects of glucocorticoid on the regulation of receptor mRNAs encoded by the mutant receptor cDNAs were examined. Deletions within the 5' half of the receptor cDNA produced transcripts that were susceptible to glucocorticoid-mediated down-regulation, whereas deletion of sequences located in the 3'-end of the receptor-coding sequence (corresponding to amino acids 550-697) resulted in receptor transcripts that were only minimally down-regulated by glucocorticoid. Together these studies indicate that multiple mechanisms control GR mRNA abundance, and an intragenic element within the ligand-binding domain is critical for this down-regulation.