The role of 11beta-hydroxysteroid dehydrogenase in steroid hormone specificity.
The role of 11beta-hydroxysteroid dehydrogenase in steroid hormone specificity.
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11β-羟基类固醇脱氢酶在类固醇激素特异性中的作用。
DOI：
10.1016/s0960-0760(98)00009-0
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发表时间：
1998
期刊：
影响因子：
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通讯作者：
Fejes-Tóth,G
中科院分区：
文献类型：
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作者：
Náray-Fejes-Tóth,A;Colombowala,IK;Fejes-Tóth,G
11β-羟基类固醇脱氢酶(11β-HSD)被认为是通过保护盐皮质激素受体(MR)不被内源性糖皮质激素占据而使醛固酮特异性作用于盐皮质激素靶细胞。在醛固酮靶细胞中存在11型β-HSD，与11型β-HSD相比，它对底物有很高的亲和力，是单向的，并且喜欢NAD作为辅因子。最近从不同物种中克隆了11个β-HSD2的编码基因，并对其基因和蛋白的细胞特异性表达进行了研究。11个β-HSD2在每个醛固酮靶组织中均有表达。Northern分析表明，兔11β-HSD2在肾脏集合管中高水平表达，在结肠中低水平表达。RT-PCR实验表明，11个β-HSD2mRNA仅在肾脏内的醛固酮靶细胞中存在。我们利用编码11β-HSD2和绿色荧光蛋白的嵌合体确定了兔11β-HSD2的亚细胞定位。将此载体稳定地导入CHO和MDCK细胞。表达的11β-HSD2/GFP蛋白保持了较高的酶活性，其性质与天然酶无明显区别。荧光显微镜观察该蛋白在细胞内的定位。11β-HSD2相关荧光在细胞质上呈网状分布，而质膜和胞核均为阴性，提示内质网(ER)的定位。与ER蛋白、高尔基体膜、线粒体和细胞核的标记共染色证实，11β-HSD2仅定位于ER。为了确定哪些结构基序负责ER的定位，我们产生了缺失C端42和118个氨基酸的缺失突变体，并将它们与GFP融合。与完整的11β-hsd2类似，这些突变体仅定位于内质网。两个C末端缺失突变体都完全失去了脱氢酶活性，无论是在完整的细胞中还是在匀浆中确定活性。这些结果表明，11β-HSD2具有一个新的ER恢复信号，该信号不局限于C-末端区域。此外，C末端的第118个氨基酸是依赖NAD的11β-HSD的必需氨基酸。
11β-hydroxysteroid dehydrogenase (11β-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. In aldosterone target cells the type 2 11β-HSD is present, which, in contrast to the type 1 11β-HSD, has very high affinity for its substrate, is unidirectional and prefers NAD as cofactor. cDNAs encoding 11β-HSD2 have been recently cloned from different species, and the cell-specific expression of its mRNA and protein were determined. 11β-HSD2 is expressed in every aldosterone target tissue. Northern analysis revealed that the rabbit 11β-HSD2 is expressed at high levels in the renal collecting duct and at much lower levels in the colon. RT-PCR experiments demonstrated that 11β-HSD2 mRNA is present only in aldosterone target cells within the kidney. We determined the subcellular localization of the rabbit 11β-HSD2 using a chimera encoding 11β-HSD2 and the green fluorescent protein (GFP). This construct was stably transfected into CHO and MDCK cells. The expressed 11β-HSD2/GFP protein retained high enzymatic activity, and its characteristics were undistinguishable from those of the native enzyme. The intracellular localization of this protein was determined by fluorescence microscopy. 11β-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Co-staining with markers for ER proteins, the Golgi membrane, mitochondria and nucleus confirmed that 11β-HSD2 is localized exclusively to the ER. To determine what structural motifs are responsible for the ER localization, we generated deletion mutants missing the C-terminal 42 and 118 amino acids, and fused them to GFP. Similarly as with the intact 11β-HSD2, these mutants localized exclusively to the ER. Both C-terminal deletion mutants completely lost dehydrogenase activity, independently whether activity was determined in intact cells or homogenates. These results indicate that 11β-HSD2 has a novel ER retrieval signal which is not localized to the C-terminal region. In addition, the C-terminal 118 amino acids are essential for NAD-dependent 11β-HSD activity.