Regulatory Roles of PNRC in Breast Epithelial Cells

PNRC 在乳腺上皮细胞中的调节作用

基本信息

批准号：
6837594
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
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-02-01 至 2008-01-31
项目状态：
已结题

项目摘要

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.

该提出的研究将检验以下假设：与GRB2（生长因子受体结合蛋白2）一起，PNRC（富含脯氨酸的核受体共调控蛋白）调节了核受体介导的调节和人类乳腺外皮细胞中介导的生长因子/RAS介导的调节。通过使用牛SF1（类固醇生成因子1）作为诱饵，在酵母的两杂交筛选中鉴定了PNRC。我们的实验室已经产生了三个重要的结果。首先，通过使用RT-PCR，发现PNRC在乳房上皮细胞中有选择地表达，并且发现非癌细胞系中的表达水平高于乳腺癌细胞系中的表达水平。其次，PNRC被证明是核受体（例如ER（雌激素受体）和PR（孕酮受体）的共激活因子。共激活因子可以被GRB2抑制。第三，通过与GRB2相互作用，PNRC抑制了生长因子诱导的MAP（有丝分裂原激活的蛋白）激酶激活。该提案有五个具体的目标。 AIM 1将通过稳定的转染实验研究PNRC的功能。 AIM 2将使用转染的荧光蛋白（FP）-PNRC和FP-GRB2融合蛋白通过实验研究PNRC和GRB2细胞内运动的动力学。 AIM 3将通过蛋白质缺失和定向诱变实验来确定PNRC与核受体之间相互作用的分子基础。 AIM 4将确定PNRC和GRB2之间相互作用的分子基础。 AIM 5将应用酵母两杂交筛选方法来鉴定与PNRC相互作用的人类乳腺组织中的蛋白质。这些实验将确定PNRC（PNRC2）和GRB2之间相互作用的分子基础，检查这些蛋白质在核受体介导的途径上的功能以及人类乳腺中的生长因子介导的途径，并确定与PNRC相互作用的乳腺组织中的其他分子。该实验室熟悉本应用程序中提出的所有实验技术。已经对核受体信号途径和生长因子信号途径都进行了广泛的研究，这些途径的控制机制很复杂。但是，PNRC和GRB2上的发现代表了一种调节这些途径的新鉴定的机制。