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首页> 外文期刊>The Journal of Steroid Biochemistry and Molecular Biology >Putative steroid binding domain of the human mineralocorticoid receptor, expressed in E. coli in the presence of heat shock proteins shows typical native receptor characteristics.
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Putative steroid binding domain of the human mineralocorticoid receptor, expressed in E. coli in the presence of heat shock proteins shows typical native receptor characteristics.

机译：在热激蛋白的存在下在大肠杆菌中表达的人盐皮质激素受体的类固醇结合域显示出典型的天然受体特征。

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摘要

Domain E, considered as the putative hormone binding domain (HBD) of the human mineralocorticoid receptor (hMR) was expressed in Escherichia coli as a fusion protein with either maltose binding protein (MBP) or glutathione S-transferase (GST). These bacterially-produced MR constructs had no steroid binding activity per se. In fact, heat shock protein association (hsp) is required for high affinity ligand-binding of the MR. After incubation of purified MBP- or GST-HBD with rabbit reticulocyte lysate, known to be rich in heat shock proteins, we obtained saturable binding of [3H]aldosterone. The Kd value for aldosterone was 0.3 nM and the Bmax = 32 pmol/mg. Hormone binding specificity was assessed by competition studies with various steroid ligands. Sucrose gradient assays performed with [3H]aldosterone-MBP-HBD revealed complex sedimenting at 8.3S and 4.9S with [3H]progesterone-MBP-HBD. Western-blot analysis of the sedimentation peak showed the concomitant presence of MBP-HBD by a monoclonal anti-MBP antibody, and hsp90 by a monoclonal anti-hsp antibody. Moreover, following incubation with the anti-rabbit hsp90 monoclonal antibody the sedimenting gradient showed a 10.4S sedimenting complex. These analyses demonstrated that the [3H]aldosterone-MBP-HBD complex is at least associated with hsp90 in reticulocyte lysate and that the HBD of hMR is sufficient to bind hsp90. Deletions of a relatively short amino- (729-766) or carboxy-terminal (940-984) region of the HBD fragment eliminated all steroid-binding properties. Overall, these results indicate that the integrity of domain E is necessary and sufficient to bind steroid ligands, agonists or antagonists, with characteristics similar to the entire native MR.
机译：域E被认为是人类盐皮质激素受体（hMR）的推定激素结合域（HBD），在大肠杆菌中以与麦芽糖结合蛋白（MBP）或谷胱甘肽S-转移酶（GST）的融合蛋白表达。这些细菌产生的MR构建体本身不具有类固醇结合活性。实际上，MR的高亲和力配体结合需要热激蛋白缔合（hsp）。将纯化的MBP-或GST-HBD与已知富含热休克蛋白的兔网织红细胞裂解液孵育后，我们获得了[3H]醛固酮的饱和结合。醛固酮的Kd值为0.3 nM，Bmax = 32 pmol / mg。通过与各种类固醇配体的竞争研究评估了激素结合特异性。用[3H]醛固酮-MBP-HBD进行的蔗糖梯度测定显示，[3H]孕酮-MBP-HBD在8.3S和4.9S时有复杂的沉淀。沉淀峰的Western印迹分析显示，单克隆抗MBP抗体同时存在MBP-HBD，单克隆抗hsp抗体同时存在hsp90。此外，在与抗兔hsp90单克隆抗体一起温育后，沉降梯度显示出10.4S的沉降复合物。这些分析表明，[3H]醛固酮-MBP-HBD复合物至少与网织红细胞裂解物中的hsp90相关，并且hMR的HBD足以结合hsp90。 HBD片段的相对较短的氨基（729-766）或羧基末端（940-984）区域的缺失消除了所有类固醇结合特性。总体而言，这些结果表明，结构域E的完整性对于结合类固醇配体，激动剂或拮抗剂具有必要和充分的作用，其特征类似于整个天然MR。

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