MINERALOCORTICOID RECEPTOR

介导信号的盐皮质激素受体(MR) 由醛固酮产生的是一种重要的受体，参与 应激机制：由于类固醇受体复合体的不稳定性 在组织提取物中，其性质和功能一直不是很好 因此，我们建议在细胞中过表达人类MR。 昆虫细胞使用杆状病毒系统。这将有助于 可纯化的毫克量受体的产生 果翅夜蛾Sf9细胞。此外，第一个350个氨基酸 截短MR和DNA结合域，但缺少配体 结合结构域将过度表达，全长GR-MR嵌合体也将过度表达 受体。这些受体将从Sf9细胞中纯化出来用于实验 与激活和转录功能有关。低聚物形式 将由过表达和纯化的MR、HSP70重组 和其他纯化的胞浆因子(P59，调节剂)研究 组装机制和确定细胞质激活机制 在试管中。MR及其衍生物的特定DNA结合将是 分析了无细胞转录的激活情况。这将是 需要特别磷酸化的MR及其不同形式 在Sf9细胞中高效表达和纯化。MR反应元件 将被映射到Na，K-ATPaseα和 通过足迹法检测β亚基的DNA。特定的转录因子 与N-末端结构域相互作用后将其结合 通过化学的(和可逆的)交叉连接到过表达的N末端。 链接。一种特定的抗MR抗体，不会与 糖皮质激素受体将从预测的多肽中产生 N-末端的序列。该抗体将用于DNA结合 实验作为一种提纯MR的手段，这些信息将对 关于从分子水平上理解逆境适应机制的思考 将适用于人类的醛固酮增多症和 高血压。

The mineralocorticoid receptor (MR) that mediates signals generated by aldosterone is an important receptor involved in the stress mechanism.Because of the lability of the steroid-receptor complex in tissue extracts, its properties and functions have not been well characterized.Therefore, it is proposed to overexpress the human MR in insect cells using the baculovirus system. This will facilitate the generation of milligram quantities of receptor that can be purified from Spodoptera frugiperda (Sf9) cells. In addition,the first 350 amino acid truncated MR together with the DNA binding domain but lacking the ligand binding domain will be overexpressed as will a full length GR-MR chimeric receptor.These receptors will be purified from Sf9 cells for experiments bearing on activation and transcriptional functions. The oligomeric form of the MR will be reconstituted from overexpressed and purified MR, HSP70 and other purified cytosolic factors (p59, modulator) to study the mechanism of assembly and to define the cytoplasmic activation mechanism in vitro. The specific DNA binding of the MR and its derivatives will be analyzed as will the activation of cell-free transcription. This will entail specifically phosphorylated MR and its different forms that have been overexpressed and purified from Sf9 cells.The MR responsive element will be mapped on the 5' flanking regions of the Na,K-ATPase alpha and beta subunits' DNA by footprinting. Specific transcription factors that interact with the N-terminal domain will be isolated after their binding to the overexpressed N-terminus by chemical (and reversible) cross- linking. A specific anti-MR antibody that will not cross-react with the glucocorticoid receptor will be generated from a predicted peptide sequence in the N-terminus. This antibody will be used in the DNA binding experiments and as a means to purify the MR. This information will impact on the understanding of the stress adaptation mechanism at the molecular level and will apply to human diseases of hyperaldosteronism and hypertension.