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/me.8.12.1764
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发表时间：
1994-12-01
影响因子：
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通讯作者：
CIDLOWSKI, JA
中科院分区：
文献类型：
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作者：
BURNSTEIN, KL;JEWELL, CM;CIDLOWSKI, JA
糖皮质激素受体 (GR) 是配体依赖性转录因子，在细胞生长、分化和死亡的内分泌控制中发挥着关键作用。人们广泛认为，这些类固醇受体在细胞培养物和动物（包括人类）中接触配体后会发生下调。细胞受体水平的降低导致对随后的激素施用不敏感。然而，人们对控制 GR 同源下调的机制知之甚少。我们之前已经证明 (1) 转染的人 OR (hGR) 互补 DNA (cDNA) 包含足以概括体内观察到的 hGR 信使 RNA (mRNA) 和蛋白质下调的序列。我们现在已经评估了涉及 hGR mRNA 激素调节的潜在机制，并且进一步鉴定了包含下调信号的 hGR cDNA 的基因内结构域。用糖皮质激素处理表达转染的 hGR cDNA 的 COS-1 细胞，在放线菌酮或放线菌素-D 存在的情况下，导致 hGR mRNA 下调，这表明糖皮质激素诱导蛋白对于下调并不是必需的。我们发现，配体延长受体占据会导致 GR mRNA 更新增加，此外，激动剂地塞米松或拮抗剂 RU486 都会降低 hGR cDNA 的转录。为了确定哪些受体 cDNA 序列对于下调至关重要，采用了共转染策略，其中一系列 hGR cDNA 缺失突变体与全长 hGR cDNA 一起转染。检查了糖皮质激素对突变受体 cDNA 编码的受体 mRNA 的调节作用。受体 cDNA 5' 半部内的删除产生的转录物易受糖皮质激素介导的下调影响，而位于受体编码序列 3' 端的序列（对应于氨基酸 550-697）的删除导致受体转录物仅被糖皮质激素最小程度地下调。这些研究共同表明，多种机制控制 GR mRNA 丰度，配体结合域内的基因内元件对于这种下调至关重要，
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 OR (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,