TISSUE-SPECIFIC EFFECTS OF PROGESTINS

描述：（改编自申请人的摘要）有两种孕酮 受体（PR）：933 aa B-受体和769 aa A-受体，164 aa N-末端截短。B：A的比例在不同的组织中不同;由B 与A相比，在配体、启动子和细胞特异性方面不同; B是 当A是抑制剂时，反式激活剂。PR N-末端含有 同源DNA结合域（DBD），但缺乏激素结合域 (HBD)是独立的功能域。它们是构成性的 反式激活因子，概括了全长的功能差异， 受体。假设：N末端的结构差异解释了 两个PR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 将NT-B加上全长A-和B-受体纯化至均一。 生化功能将通过体外转录、DNA结合 亲和性和协同性将被量化;自缔合特性将 通过沉降平衡进行分析;尺寸和形状由 沉降速度纯化的N-末端的结构将通过 有限的蛋白水解和光谱分析。DNA引起的构象变化 结合和HBD将被映射。这些研究将定义结构 在两个PR的N-末端处的差异。AIM 2.分离与 介导的独特功能特性的蛋白质的表征 两个PR N-末端。我们假设N-末端的结构差异 导致独特转录辅助调节因子募集到每个PR同种型。 NT-A和NT-B将在酵母单杂交和双杂交测定中用作"诱饵"， 传统的层析蛋白分离和测序， 与两个PR N-末端，或那些只与一个相互作用，并赋予独特的 每种PR亚型的特性。AIM 3.突变和转录分析 NT-A和NT-B的结构亚域。初步数据显示，NT-A是 组织成7个蛋白酶不可接近的盒，由6个溶剂暴露的 环，其激活功能是一个稳定的亚基。这些领域，以及 NT-B中新定义的结构和功能结构域将突变为 评估它们在NT-A与NT-B组成型转录中的作用，以及 配体调控的转录全长PR。拟议的研究地址 两个PR N-末端目前未知的功能。

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.