CAMP SIGNALING AND GENE TRANSCRIPTION IN HAPLOID CELLS

单倍体细胞中的 CAMP 信号传导和基因转录

• 批准号: 2205597
• 负责人: MICHAEL W COLLARD
• 金额: $ 9.19万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2205597
• 关键词: RNase protection assay; androgen receptor; biological signal transduction; cyclic AMP; enzyme activity; gel mobility shift assay; genetic promoter element; genetic transcription; genetically modified animals; germ cells; haploidy; immunocytochemistry; in situ hybridization; laboratory mouse; laboratory rat; messenger RNA; northern blottings; nucleic acid sequence; nucleoproteins; protein isoforms; protein kinase A; site directed mutagenesis; testis; tissue /cell culture; transcription factor

Cyclic-AMP-dependent protein kinase (PKA) phosphorylates serine and threonine residues of substrate proteins to modify cellular functions such as gene expression and cell cycle progression. The RIIalpha regulatory subunit of PKA is expressed at elevated levels in differentiating haploid germinal cells and occupies a central role in cAMP signaling during mammalian spermatogenesis. In spermatozoa, RIIalpha is associated with the flagella and regulates cAMP-dependent activation of sperm cell motility. In earlier steps of germ cell development, non-flagellar RIIalpha is associated with the nucleus and will regulate PKAdependent phosphorylation of haploid specific transcription factors such as CREMtau. Activation of nuclear PKA could be the genetic switch which leads to broad expression of genes in haploid cells. Two germ cell specific mRNA isoforms for RIIalpha are observed to be differentially regulated in stage- synchronized testis. We hypothesize that the RIIalpha mRNA isoforms represent flagellar and cytoplasmic/nuclear protein isoforms required for appropriate sub cellular localization and propose to (i) characterize the structural (sequence) differences between the two RIIalpha isoforms and to identify corresponding germ cell specific RIIalpha A-kinase anchoring proteins (AKAPs) and (ii) to confirm the precise stage specific regulation of RIIalpha isoforms in testis. Using the 5' promoter region of the RIIalpha gene and transient transfection assays, we have identified a DNA regulatory region which is responsible for androgen activation and transcriptional feedback inhibition by PKA. This novel region of DNA also interacts with germ cell nuclear proteins. We propose to study signal transduction crosstalk between the cAMP and androgen signaling pathways through (iii) transient transfection of transcriptional reporter genes, PKA expression vectors, and cloned transcription factors into permissive cell lines, and to characterize the germ cell nuclear proteins which bind to promoter sequences. We also propose to (iv) analyze RIIalpha promoter regulation in developing germ cells using transgenic mice. The long term goal of this research is to provide a foundation for understanding male infertility problems related to motility or abnormal germ cell development, and to provide basic information on androgen and cAMP signal transduction crosstalk mechanisms used by normal and neoplastic tissues.

环磷酸腺苷依赖的蛋白激酶(PKA)磷酸化丝氨酸和 底物蛋白的苏氨酸残基可以改变细胞功能，如 作为基因表达和细胞周期进程。RIIpha监管机构 PKA亚单位在单倍体分化中的高水平表达 生发细胞在cAMP信号转导中起中心作用 哺乳动物的精子发生。在精子中，RIIpha与 鞭毛和调节cAMP依赖的精子细胞的激活 能动性。在生殖细胞发育的早期阶段，非鞭毛 RIIpha与核相关，并将调节PKA依赖 单倍体特异转录因子如CREMtau的磷酸化。 核PKA的激活可能是导致广泛的 基因在单倍体细胞中的表达。两种生殖细胞特异的mRNA异构体 对于RIIpha，观察到在不同阶段受到不同的调节- 同步的睾丸。我们假设RIIpha信使核糖核酸亚型 代表鞭毛和细胞质/核蛋白亚型 适当的亚细胞定位，并建议(I)表征 两种RIIpha亚型和TO亚型的结构(序列)差异 确定相应的生殖细胞特异性RIIAlphaA-激酶锚定 蛋白质(AKAPs)和(Ii)以确认精确的阶段特异性调控 在睾丸中的RIIAlpha亚型。利用该基因的5‘端启动子区 RIIpha基因和瞬时转染实验，我们已经鉴定出一种DNA 负责雄激素激活和 PKA对转录反馈的抑制作用。这个新的DNA区域也 与生殖细胞核蛋白相互作用。我们建议研究信号 CAMP和雄激素信号通路之间的信号转导串扰 通过(Iii)转录报告基因的瞬时转染， PKA表达载体，并将转录因子克隆到允许的 细胞系，并鉴定与生殖细胞核蛋白结合的 到启动子序列。我们还建议(Iv)分析RIIpha启动子 转基因小鼠发育生殖细胞的调控。从长远来看 本研究的目的是为理解男性提供一个基础 与运动或生殖细胞异常有关的不孕问题 发展，并提供有关雄激素和cAMP信号的基本信息 正常组织和肿瘤组织使用的转导串扰机制。