Regulatory Roles of PNRC in Breast Epithelial Cells

PNRC 在乳腺上皮细胞中的调节作用

• 批准号: 6620436
• 负责人: Shiuan Chen
• 金额: $ 27.56万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6620436
• 关键词: MCF7 cell; biological signal transduction; cell proliferation; chimeric proteins; enzyme activity; estrogen receptors; growth factor receptors; human tissue; intracellular transport; mammary epithelium; mammary gland; mitogen activated protein kinase; nuclear receptors; polymerase chain reaction; progesterone receptors; protein protein interaction; protein structure function; receptor binding; site directed mutagenesis; transfection /expression vector; yeast two hybrid system

This proposed investigation will test the hypothesis that, together with Grb2 (Growth factor receptor bound protein- 2), PNRC (Proline-rich Nuclear Receptor Co-regulatory protein) modulates both the nuclear receptor-mediated regulation and the growth factor/Ras mediated regulation in human breast epithelial cells. PNRC was identified in a yeast two-hybrid screening of a human mammary gland cDNA expression library by using bovine SF1 (Steroidogenic Factor 1) as bait. Three important sets of results have been generated in our laboratory. First, by using RT-PCR, PNRC was found to be selectively expressed in breast epithelial cells, and the expression levels in non-cancerous cell lines were found to be higher than those in breast cancer cell lines. Second, PNRC was shown to act as a coactivator for nuclear receptors such as ER (estrogen receptor) and PR (progesterone receptor). The coactivator activity can be suppressed by Grb2. Third, by interacting with Grb2, PNRC suppresses growth factor-induced MAP (mitogen-activated protein) kinase activation. There are five specific aims in this proposal. Aim 1 will study the function of PNRC by stable transfection experiments. Aim 2 will study the dynamics of intracellular movement of PNRC and Grb2 with experiments using transfected fluorescent protein (FP)-PNRC and FP-Grb2 fusion proteins. Aim 3 will determine the molecular basis of the interaction between PNRC and nuclear receptors by protein deletion and site-directed mutagenesis experiments. Aim 4 will determine the molecular basis of the interaction between PNRC and Grb2. Aim 5 will apply the yeast two-hybrid screening method to identify protein(s) in human breast tissue that interacts with PNRC. These experiments will determine the molecular basis of the interaction between PNRC (PNRC2) and Grb2, examine the functions of these proteins on the nuclear receptor-mediated pathway and the growth factor-mediated pathway in human breast, and identify additional molecules in breast tissue that interact with PNRC. This laboratory is familiar with all the experimental techniques proposed in this application. Both the nuclear receptor-signaling pathway and the growth factor-signaling pathway have been extensively studied, and the control mechanisms for these pathways are complex. However, the findings on PNRC and Grb2 represent a newly identified mechanism for modulating these pathways.

本研究将验证一个假设，即PNRC（富含脯氨酸的核受体共调节蛋白）与Grb2（生长因子受体结合蛋白- 2）一起调节人类乳腺上皮细胞中核受体介导的调节和生长因子/Ras介导的调节。以牛甾体生成因子1 （Steroidogenic Factor 1, SF1）为诱饵，对人乳腺cDNA表达文库进行酵母双杂交筛选，鉴定出PNRC。我们实验室产生了三组重要的结果。首先，通过RT-PCR发现PNRC在乳腺上皮细胞中选择性表达，且在非癌细胞系中的表达水平高于乳腺癌细胞系。其次，PNRC被证明是核受体如ER（雌激素受体）和PR（孕激素受体）的辅激活剂。Grb2可以抑制共激活子的活性。第三，通过与Grb2相互作用，PNRC抑制生长因子诱导的MAP（丝裂原活化蛋白）激酶的激活。这项建议有五个具体目标。目的1通过稳定转染实验研究PNRC的功能。目的2将通过转染荧光蛋白(FP)-PNRC和FP-Grb2融合蛋白的实验，研究PNRC和Grb2的胞内运动动力学。目的3将通过蛋白缺失和定点诱变实验确定PNRC与核受体相互作用的分子基础。目的4将确定PNRC与Grb2相互作用的分子基础。目标5将应用酵母双杂交筛选方法来鉴定人类乳腺组织中与PNRC相互作用的蛋白质。这些实验将确定PNRC （PNRC2）和Grb2相互作用的分子基础，研究这些蛋白在人类乳腺中核受体介导途径和生长因子介导途径中的功能，并确定乳腺组织中与PNRC相互作用的其他分子。本实验室熟悉本申请中提出的所有实验技术。核受体-信号通路和生长因子-信号通路均已被广泛研究，其调控机制复杂。然而，PNRC和Grb2的研究结果代表了一种新发现的调节这些途径的机制。