Regulatory Roles of PNRC in Breast Epithelial Cells

这项拟议的研究将验证这样的假设，即富含Pro的核受体共调节蛋白PNRC与生长因子受体结合蛋白-2(Grb2)一起调节人乳腺上皮细胞的核受体调节和生长因子/RAS调节。以牛类固醇生成因子1为诱饵，在人乳腺表达文库的酵母双杂交筛选中鉴定到PNRC。我们的实验室已经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，并且在非癌细胞系中的表达水平高于乳腺癌细胞系。其次，PNRC被证明是雌激素受体(ER)和孕激素受体(PR)等核受体的共同激活剂。共激活子活性可被Grb2抑制。第三，通过与Grb2的相互作用，PNRC抑制生长因子诱导的MAP(丝裂原活化蛋白)激酶的激活。这项提议有五个具体目标。目的1通过稳定转染实验研究PNRC的功能。目的2利用转基因荧光蛋白(FP)-PNRC和FP-Grb2融合蛋白研究PNRC和Grb2在细胞内的运动动力学。目的3通过蛋白质缺失和定点突变实验，确定PNRC与核受体相互作用的分子基础。目的4将确定PNRC和Grb2相互作用的分子基础。目的5应用酵母双杂交筛选方法在人乳腺组织中鉴定与PNRC相互作用的蛋白(S)。这些实验将确定PNRC(PNRC2)和Grb2相互作用的分子基础，检测这些蛋白质在人类乳房中核受体介导的途径和生长因子介导的途径上的功能，并确定乳腺组织中与PNRC相互作用的额外分子。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-信号通路都得到了广泛的研究，而且这些通路的调控机制很复杂。然而，关于PNRC和Grb2的发现代表了一种新发现的调节这些通路的机制。

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.