Domains regulating transcriptional activity of the inducible orphan receptor NGFI-B.
Domains regulating transcriptional activity of the inducible orphan receptor NGFI-B.
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DOI：
10.1016/s0021-9258(18)42029-7
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发表时间：
1992-08
期刊：
影响因子：
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通讯作者：
R. E. Paulsen;C. Weaver;T. Fahrner;Jeffrey Milbrandt
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文献类型：
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作者：
R. E. Paulsen;C. Weaver;T. Fahrner;Jeffrey Milbrandt
NGFI-B 是一种早期反应基因，编码的蛋白质与 DNA 结合域和负责配体结合和转录活性调节的羧基末端域中的核受体具有很强的同源性。此前，我们已经证明，在没有外源添加配体的情况下，NGFI-B 在体外生长的细胞中具有转录活性。然而，NGFI-B 的配体似乎不是细胞培养基的成分，因为当在缺乏酚红、血清、必需维生素或必需氨基酸的培养基中生长的细胞中表达时，NGFI-B 仍保持活性。为了定义反式激活结构域，进行了突变分析，结果表明氨基末端内富含丝氨酸/苏氨酸的区域（称为 TAB-1），富含 18 个氨基酸，是一个重要的转录激活结构域。 TAB-1 内两个相邻丝氨酸和苏氨酸残基的突变显着降低了 NGFI-B 的反式激活。对羧基末端在调节 NGFI-B 转录活性中的作用的检查表明，与其他核受体一样，缺乏羧基末端部分的突变体的活性大大降低。突变体 B delta 414-597 的研究进一步支持了与其他受体的相似性，该突变体编码完全活性的截短受体，类似于激素独立的、组成性活性糖皮质激素受体截短突变体。这种 NGFI-B 截短突变体在许多哺乳动物细胞系中具有与野生型 NGFI-B 相似的活性；然而，相比之下，它的活性比果蝇 S2 细胞系中的野生型受体高 8 倍，这表明昆虫细胞要么缺乏 NGFI-B 配体，要么缺乏必需的辅助因子。
NGFI-B is an early response gene which encodes a protein that has strong homology with nuclear receptors in the DNA binding domain and in carboxyl-terminal domains responsible for ligand binding and regulation of transcriptional activity. Previously, we have demonstrated that NGFI-B is transcriptionally active in cells grown in vitro in the absence of exogenously added ligand. However, the ligand for NGFI-B does not appear to be a component of cell culture medium, as NGFI-B remained active when expressed in cells grown in medium lacking phenol red, serum, essential vitamins, or essential amino acids. To define the transactivation domains, a mutational analysis was conducted which revealed that a serine/threonine-rich area of 18 amino acids within the amino terminus, termed TAB-1, is an important transcriptional activation domain. The mutation of two adjacent serine and threonine residues within TAB-1 significantly decreased transactivation by NGFI-B. An examination of the role of the carboxyl terminus in regulating NGFI-B transcriptional activity revealed that, in accordance with other nuclear receptors, mutants lacking portions of the carboxyl terminus had greatly decreased activity. The similarity with other receptors was further supported by studies with the mutant B delta 414-597 which encodes a fully active, truncated receptor analogous to a hormone-independent, constitutively active glucocorticoid receptor truncation mutant. This NGFI-B truncation mutant had activity similar to wild type NGFI-B in a number of mammalian cell lines; however, in contrast, it was 8-fold more active than the wild type receptor in the Drosophila S2 cell line, suggesting that insect cells either lack the NGFI-B ligand or obligatory accessory factors.