GLUCOCORTICOID RECEPTOR STRUCTURE: SUBUNIT ARCITECTURE

糖皮质激素受体结构：亚基结构

• 批准号: 3235443
• 负责人: ROBERT W HARRISON
• 金额: $ 9.88万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1987-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3235443
• 关键词: affinity chromatography; complementary DNA; glucocorticoids

A panel of hybridomas producing monoclonal antibodies to the rat glucocorticoid receptor has been produced, and four stable clones from that panel have already been established. Two of the stable clones, BuGR1 and BuGR2, produce antibodies recognizing different epitopes on the receptor. BuGR1 antibody has been successfully used by us to perform immunoaffinity chromatography of the glucocorticoid receptor and by a collaborator to identify an expression vector containing a recombinant clone of rat cDNA which produces a peptide containing the BuGR1 epitope. We propose to: 1) develop stable hybridoma clones producing antibodies to epitopes located in the steroid-binding and DNA-binding regions of the receptor; 2) purify the activated and native forms of the receptor by conventional and immunoaffinity techniques, and; 3) determine the subunit, multimeric composition of the receptor. Individual hybridoma clones will be screened for their ability to detect the meroreceptor, for their ability to block steroid binding to the receptor, and for their ability to block receptor binding to DNA. To purify the activated and native forms of the receptor, DNA cellulose chromatography (for the activated receptor) or steroid-affinity chromatography (for the native receptor) will be followed by immunoaffinity chromatography. A peptide containing the antibody epitope will be used to competitively elute the receptor from the immunoaffinity column in an undenatured form. The multimeric structure of the immunopurified, denatured receptor will be studied by classical bifunctional cross-linking methods. Ultimately, the fruits of this project will be a detailed description of the structural features of the rat glucocorticoid receptor, including its amino acid sequence, and an understanding of the relationship of those features to biologically important functions of the glucocorticoid receptor.

一组产生抗大鼠单克隆抗体的杂交瘤 已经产生了糖皮质激素受体，并从中产生了四个稳定的克隆， 小组已经成立。 两个稳定的克隆，BuGR 1和 BuGR 2产生识别受体上不同表位的抗体。 BuGR 1抗体已被我们成功地用于进行免疫亲和 糖皮质激素受体的色谱法，并由合作者， 鉴定含有大鼠cDNA重组克隆的表达载体 其产生含有BuGR 1表位的肽。 我们建议：1） 开发稳定的杂交瘤克隆，其产生针对位于 受体的类固醇结合区和DNA结合区; 2）纯化 通过常规的和 免疫亲和技术，和; 3）确定亚基，多聚体 受体的组成。 将筛选单个杂交瘤克隆 因为它们能够检测局部感受器， 类固醇与受体的结合，以及它们阻断受体的能力 与DNA结合。 为了纯化受体的活化形式和天然形式， DNA纤维素层析（用于活化受体）或 将遵循类固醇亲和色谱法（用于天然受体 通过免疫亲和层析。 含有抗体的肽 表位将被用于竞争性地将受体从 免疫亲和柱。 的多聚体结构 免疫纯化的变性受体将通过经典的 双功能交联方法。 最终，这个项目的成果 将详细描述大鼠的结构特征 糖皮质激素受体，包括其氨基酸序列，和 了解这些特征与生物学的关系， 糖皮质激素受体的重要功能。