TISSUE-SPECIFIC EFFECTS OF PROGESTINS

描述：(改编自申请人的摘要)有两种黄体酮 受体(PR)：933个氨基酸B受体和769个氨基酸A受体，其中164个氨基酸 N端截断。B：A的比例因组织不同而不同； A和A在配体、启动子和细胞特定的方式上不同；B是 反式激活剂当A是抑制物时。PR N-末端包含 同源DNA结合域(DBD)，但缺少激素结合域 (HBD)，是独立的功能结构域。它们是构成要素 反式激活剂，概括了全长的功能差异 感受器。假说：N端结构差异的解释 两种公关的功能差异。目的1.A-(NT-A)的结构和 B受体(NT-B)N-末端。功能自治域、NT-A和 NT-B，加上全长的A和B受体，将被提纯为均一。 生化功能将通过体外转录、DNA结合来证明 亲和力和协作性将被量化；自关联属性将被量化 根据沉积平衡进行分析；大小和形状由 沉积速度。纯化的N-末端的结构将通过 有限的蛋白质分解和光谱分析。DNA引起的构象变化 绑定和HBD将被映射。这些研究将定义结构 两个PR的N-末端的差异。目标2.隔离和 介导细胞独特功能特性的蛋白质的特性 两个PR N-末端。我们假设N-末端的结构差异 导致每个PR亚型招募独特的转录协同调节因子。 NT-A和NT-B将在酵母单杂交和双杂交试验中用作“诱饵”，并在 相互作用的常规蛋白质层析分离和测序 既有PR N-末端，也有只与一个相互作用并赋予唯一的 属性添加到每个PR异构体。目标3.突变和转录分析 NT-A与NT-B的结构亚域的比例。初步数据显示，NT-A是 组织成7个不能接触到蛋白酶的盒，由6个暴露在溶剂中的隔开 它的激活功能是一个稳定的亚基。这些域，以及 NT-B中新定义的结构和功能结构域将突变为 通过NT-A和NT-B评估它们在构成转录中的作用，以及 配体调控的全长PR转录。拟议的研究旨在解决 目前尚不清楚两个PR N末端的功能。

DESCRIPTION: (Adapted from the applicant's abstract) There are two progesterone receptors (PR): 933 aa B-receptors, and 769 aa A-receptors with a 164 aa N-terminal truncation. The B:A ratio differs among tissues; transcription by B vs. A differs in ligand-, promoter- and cell-specific ways; B are transactivators when A are inhibitors. The PR N-termini containing the homologous DNA binding domain (DBD) but lacking the hormone binding domain (HBD), are independent functional domains. They are constitutive transactivators that recapitulate the functional differences of the full-length receptors. Hypothesis: structural differences at their N-termini explain functional differences of the two PR. Aim 1. Structure of A- (NT-A) and B-receptor (NT-B) N-termini. The functionally autonomous domains, NT-A and NT-B, plus full-length A- and B- receptors, will be purified to homogeneity. Biochemical function will be documented by in vitro transcription, DNA binding affinity and cooperativity will be quantified; self-association properties will be analyzed by sedimentation equilibrium; size and shape defined by sedimentation velocity. The structure of purified N-termini will be compared by limited proteolysis and spectroscopic assays. Conformational changes due to DNA binding and the HBD will be mapped. These studies will define structural differences at the N-termini of the two PR. AIM 2. Isolation and characterization of proteins that mediate the unique functional properties of the two PR N-termini. We postulate that structural differences at the N-termini lead to recruitment of unique transcriptional co-regulators to each PR isoform. NT-A and NT-B will be used as "bait" in yeast one-and two- hybrid assays and in conventional chromatographic protein isolation and sequencing that interact with both PR N-termini, or ones that interact only with one, and confer unique properties to each PR isoform. AIM 3. Mutational and transcriptional analyses of structural subdomains in NT-A vs. NT-B. Preliminary data show that NT-A is organized into 7 protease inaccessible cassettes separated by 6 solvent-exposed loops and that its activation function is a stable subunit. These domains, and newly defined structural and functional domains in NT-B, will be mutated to assess their role in constitutive transcription by NT-A vs. NT-B, and ligand-regulated transcription by full-length PR. The proposed studies address the presently unknown functions of the two PR N-termini.