Jaglaguier S, Mesnier D, Láger J L, Auzou G
Institut National de la Santé et de la Recherche Médicale, Faculté de Pharmacie, Montpellier, France.
J Steroid Biochem Mol Biol. 1996 Jan;57(1-2):43-50. doi: 10.1016/0960-0760(95)00250-2.
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.
结构域E被认为是人类盐皮质激素受体（hMR）的假定激素结合结构域（HBD），它在大肠杆菌中作为与麦芽糖结合蛋白（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构建体本身没有类固醇结合活性。事实上，热休克蛋白结合（hsp）是MR高亲和力配体结合所必需的。在用已知富含热休克蛋白的兔网织红细胞裂解物孵育纯化的MBP-或GST-HBD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激素结合特异性。用[3H]醛固酮-MBP-HBD进行的蔗糖梯度分析显示，与[3H]孕酮-MBP-HBD一起在8.3S和4.9S处有复合物沉淀。沉淀峰的蛋白质免疫印迹分析显示，通过单克隆抗-MBP抗体可同时检测到MBP-HBD，通过单克隆抗-hsp抗体可检测到hsp90。此外，在用抗兔hsp90单克隆抗体孵育后，沉淀梯度显示出10.4S的沉淀复合物。这些分析表明，[3H]醛固酮-MBP-HBD复合物在网织红细胞裂解物中至少与hsp90相关，并且hMR的HBD足以结合hsp90。HBD片段相对较短的氨基末端（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、激动剂或拮抗剂是必要且充分的，其特性与整个天然MR相似。
