Modulation of human mineralocorticoid receptor function by protein kinase A.
Modulation of human mineralocorticoid receptor function by protein kinase A.
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蛋白激酶 A 调节人盐皮质激素受体功能。
作者：
C. Massaad;Nathalie Houard;Marc Lombès;R. Barouki
盐皮质激素受体（MR）作为一种配体依赖性转录因子，调节钠转运上皮细胞中的特异性基因表达。生理学证据表明cAMP和醛固酮信号通路之间存在串扰。我们提供的证据表明，蛋白激酶A（PKA），信号转导通路的主要介质，调节人类MR（hMR）的转录活性。使用瞬时转染实验在HepG2细胞中，我们表明，8-溴-cAMP，蛋白激酶A激活剂，刺激糖皮质激素反应元件（GRE）的启动子在配体独立的方式。这种效应是严格的MR依赖性，因为没有激活的报告基因，观察到在没有共转染的hMR表达质粒。此外，当用醛固酮和cAMP处理细胞时，实现了协同活化。在CV1和稳定表达hMR的M细胞中也观察到这种协同效应，但依赖于所用的启动子。特别是，在含有几个GRES的启动子中协同作用不太明显。我们发现，蛋白激酶抑制肽（PKI），PKA的肽抑制剂，阻止cAMP和醛固酮的诱导，这表明一个功能性的cAMP途径是必需的醛固酮刺激转录。使用MR富集的杆状病毒提取物在凝胶迁移试验中，我们已经表明，MR的结合到一个含有GRE的寡核苷酸增强PKA。通过Scatchard分析，hMR的DNA结合增加可能反映了活性受体数量的增加。使用截断MR，我们表明，N-末端域的效果是必需的。最后，N-末端截短的MR在体外不被PKA直接磷酸化。我们的结论是PKA的行为间接，可能是通过减轻MR抑制剂的影响。
The mineralocorticoid receptor (MR) acts as a ligand-dependent transcription factor modulating specific gene expression in sodium-transporting epithelia. Physiological evidence suggest a cross-talk between the cAMP- and aldosterone-signaling pathways. We provide evidence that protein kinase A (PKA), a major mediator of signal transduction pathways, modulates transcriptional activity of the human MR (hMR). Using transient transfection assays in HepG2 cells, we show that 8-bromo-cAMP, a protein kinase A activator, stimulates glucocorticoid response element (GRE)-containing promoters in a ligand-independent manner. This effect was strictly MR dependent since no activation of the reporter gene was observed in the absence of cotransfected hMR expression plasmid. Furthermore, a synergistic activation was achieved when cells were treated with both aldosterone and cAMP. This synergistic effect was also observed in the CV1 and the stable hMR-expressing M cells but was dependent on the promoter used. In particular, synergism was less pronounced in promoters containing several GREs. We show that (protein kinase-inhibiting peptide (PKI), the peptide inhibitor of PKA, prevented both cAMP and aldosterone induction, which indicates that a functional cAMP pathway is required for stimulation of transcription by aldosterone. Using MR-enriched baculovirus extracts in gel shift assays, we have shown that the binding of the MR to a GRE-containing oligonucleotide was enhanced by PKA. Increased DNA binding of hMR is likely to reflect an increase in the number of active receptors, as measured by Scatchard analysis. Using a truncated MR, we show that the N-terminal domain is required for the effect. Finally, the N-terminal truncated MR was not directly phosphorylated by PKA in vitro. We conclude that PKA acts indirectly, probably by relieving the effect of an MR repressor.
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DOI：
10.1073/pnas.86.13.4887
发表时间：
1989-07-01
影响因子：
11.1
作者：
MELLON, PL;CLEGG, CH;MCKNIGHT, GS
通讯作者：
MCKNIGHT, GS
雌二醇和蛋白激酶激活剂协同激活雌激素受体介导的转录。
DOI：
10.1210/mend.7.3.7683375
发表时间：
1993
期刊：
Molecular endocrinology (Baltimore, Md.)
影响因子：
--
作者：
Cho,H;Katzenellenbogen,BS
通讯作者：
Katzenellenbogen,BS
磷酸化和类固醇激素作用。
影响因子：
--
作者：
Bai,W;Weigel,NL
通讯作者：
Weigel,NL
生理浓度的 AVP 和醛固酮对大鼠 CCD 中的 Na 转运具有协同作用。
DOI：
--
发表时间：
1996
期刊：
Kidney international. Supplement.
影响因子：
--
作者：
Hawk,CT;Li,L;Schafer,JA
通讯作者：
Schafer,JA
cAMP 依赖性蛋白激酶热稳定抑制剂在大肠杆菌中的表达和表征。
DOI：
--
发表时间：
1991
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Thomas,J;VanPatten,SM;Howard,P;Day,KH;Mitchell,RD;Sosnick,T;Trewhella,J;Walsh,DA;Maurer,RA
通讯作者：
Maurer,RA