MINERALOCORTICOID RECEPTOR

盐皮质激素受体（MR）介导的信号 醛固酮是一种重要的受体， 由于类固醇-受体复合物的不稳定性， 在组织提取物中，其性质和功能尚未得到很好的研究 因此，提出了在哺乳动物中过表达人MR。 昆虫细胞的杆状病毒系统。这将有利于 产生毫克量的受体， 草地贪夜蛾（Spodoptera frugiperda）（Sf 9）细胞。此外，前350个氨基酸 截短的MR连同DNA结合结构域但缺乏配体 结合结构域将过表达，全长GR-MR嵌合体也将过表达 这些受体将从Sf 9细胞中纯化用于实验 与激活和转录功能有关。寡聚体形式 的MR将从过表达和纯化的MR，HSP 70 和其他纯化的胞质因子（p59，调节剂），以研究 组装机制，并确定细胞质激活机制 体外MR及其衍生物的特异性DNA结合将是 分析无细胞转录的激活。这将 需要特异性磷酸化的MR及其不同形式， 从Sf 9细胞中过表达并纯化。 将被定位在Na，K-ATP酶α的5'侧翼区域， β亚单位的DNA足迹。特异性转录因子， 与N-末端结构域相互作用的蛋白质在结合后将被分离 通过化学（和可逆）交叉连接到过表达的N-末端， 链接。一种特异性抗MR抗体，不会与 糖皮质激素受体将从预测的肽产生 在N-末端的序列。该抗体将用于DNA结合 实验，并作为一种手段，以净化MR。这一信息将影响 在分子水平上对胁迫适应机制的理解 水平，并将适用于人类疾病的醛固酮增多症， 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.