GENE SEQUENCES INVOLVED IN PROLACTIN ACTION

这项建议的长期目标是了解催乳素是如何 催乳素(PRL)调节细胞的增殖和分化。学习 PRL刺激的有丝分裂途径上的多种成分，我们 人催乳素受体(PRL-R)的克隆与鉴定 造血素/细胞因子受体超家族和一组早期 反应基因(转录因子、细胞因子、细胞因子受体) 以及延迟的早期反应基因，如RNUDC。它的结构和 其中两个基因的功能将被分析：PRL-R，它 介导PRL和RNUDC的有丝分裂作用，其合成是一种 PRL刺激细胞增殖的结果。目标1分析 PRL-R结构域参与与信号蛋白的相互作用。怎么了？ PRL-R胞内结构域酪氨酸残基作为对接位点 为了招募转录因子STAT1和STAT5 通过诱变、转染、凝胶筛选和体外相互作用进行检测 化验。PRL-R与酶2‘，5-相互作用的意义 寡腺苷合成酶(OAS)，被鉴定为PRL-R 通过酵母双杂交遗传学相互作用的蛋白质，将被解决。 PRL-R截断将用于确定PRL-R/OAS的位置 交互作用，以及OAS正义和反义结构将用于 寻址潜在的OAS在PRL-R信号转导中的作用。目标数 2阐述了RNUDC的结构和功能。遗传互补 对Nidulans曲霉的研究表明，大鼠RNUDC是一种 真菌中核运动蛋白的功能同源物； 然而，它在哺乳动物细胞中的功能尚不清楚。我们的本地化 对中心体/高尔基体区域的RNUDC染色表明 RNUDC在中心体/高尔基体结构中发挥作用的可能性 和/或功能。一系列生化、药理、遗传和 免疫荧光显微镜研究概述以确定 RNUDC的细胞定位及其潜在功能 在哺乳动物细胞中通过正义和反义策略。最后， 50 kDa RNUDC相互作用蛋白的鉴定将由 微测序。这些合并的研究应该会提供新的见解 信号机制、信号分子和基因序列 参与PRL对细胞增殖的调节。在更广泛的范围内， 这些研究可能阐明控制细胞生长的重要步骤。 并提供了对神经内分泌-免疫系统相互作用的见解。

The long term objective of this proposal is to understand how prolactin (PRL) regulates cell proliferation and differentiation. To study multiple components along the PRL-stimulated mitogenic pathway, we cloned and characterized the PRL receptor (PRL-R), a member of the hematopoietin/cytokine receptor superfamily, and a panel of early response genes (transcription factors, cytokines, cytokine receptors) and delayed early response genes, such as RNUDC. The structure and function of two of these genes will be analyzed: the PRL-R which mediates the mitogenic effects of PRL, and RNUDC, whose synthesis is a result of PRL-stimulated cellular proliferation. AIM number 1 analyzes PRL-R domains involved in interacting with signaling proteins. How the PRL-R intracellular domain tyrosine residues serve as docking sites for recruiting the transcription factors Stat1 and Stat5 will be examined by mutagenesis, transfection, gel sift and in vitro interaction assays. The significance of PRL-R interaction with the enzyme 2',5- oligoadenylate synthetase (OAS), which was identified as a PRL-R interacting protein by yeast two-hybrid genetics, will be addressed. PRL-R truncations will be used to determine the site of PRL-R/OAS interaction, and OAS sense and anti-sense constructs will be used to address potential OAS function in PRL-R signal transduction. AIM number 2 addresses RNUDC structure and function. Genetic complementation studies in Aspergillus nidulans have shown that rat RNUDC is a functional homologue of a nuclear movement protein in the fungus; however, its function in mammalian cells is unclear. Our localization of RNUDC staining to the centrosome/Golgi region suggests the exciting possibility that RNUDC may play a role in centrosome/Golgi structure and/or function. A series of biochemical, pharmacological, genetic and immunofluorescence microscopy studies are outlined to determine the cellular localization of RNUDC, and to address potential RNUDC function by sense and anti-sense strategies in mammalian cells. Finally, the identity of a 50 kDa RNUDC interacting protein will be determined by microsequencing. These combined studies should provide new insights into the signaling mechanisms, signaling molecules, and gene sequences involved in PRL regulation of cell proliferation. On a broader scale, these studies may elucidate important steps in cellular growth controls and provide insights into neuroendocrine-immune system interactions.