MODE OF ACTION OF STEROID HORMONES

类固醇激素的作用方式

• 批准号: 3224392
• 负责人: ALLAN U MUNCK
• 金额: $ 34.69万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-09-30 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3224392
• 关键词: DNA footprinting; antiinflammatory agents; enzyme substrate; genetic transcription; glucocorticoids; high performance liquid chromatography; hormone regulation /control mechanism; immunosuppression; interferon gamma; interleukin 2; laboratory mouse; lymphokines; messenger RNA; neoplastic cell culture for noncancer research; nuclear runoff assay; phosphatase inhibitor; phosphopeptides; phosphorus; phosphorylation; protein kinase; protein structure function; radionuclides; radiotracer; site directed mutagenesis; steroid hormone receptor; tissue /cell culture; transfection

The broad objectives of the proposed research are to understand at molecular and cellular levels the physiological actions of glucocorticoids, particularly those on cells of the immune system. These actions account for widespread clinical applications of glucocorticoids as antiinflammatory and immunosuppressive agents and in treatment of lymphocytic leukemias and lymphomas. Studies will be undertaken to elucidate (1) the structure, generation and function of various phosphorylated states of glucocorticoid receptors in cells; (2) mechanisms of glucocorticoid inhibition of lymphokine production. (1) As shown in the P. I.'s laboratory with WEHI-7 mouse thymoma cells, the average number of phosphates per receptor steroid-binding protein is about 3 without hormone, increases to about 5 with hormone, and decreases to 3 or less in the saltunextractable nuclear fraction. "Null receptors", formed in ATP-depleted cells, lose one phosphate. Partial proteolysis reveals phosphoserines in the steroid-binding and N-terminal domains, but not in the DNA-binding domain. The Specific Aims are to: (a) Identify, by studies of the kinetics of phosphorylation, the receptor species that are substrates for phosphorylation. Initial results suggest the substrate for hormone-induced phosphorylation is the activated receptor. (b) Locate the phosphoaminoacids by partial sequencing of phosphopeptides obtained by HPLC analysis of limit tryptic digests. Preliminary HPLC analysis with unliganded receptors reveals two major phosphopeptides. (c) Analyze the influence of phosphorylation on biological activity using site-directed mutagenesis and kinase and phosphatase inhibitors. (d) Identify kinases that phosphorylate the receptor, using kinase-deficient cells, kinase and phosphatase inhibitors, and synthetic peptide substrates. Evidence so far indicates A-kinase is not involved. (2) Glucocorticoid immunosuppression may be due mainly to inhibition of cytokine production, which for interleukin-2 (IL-2) and gamma-interferon is associated with lowered mRNA levels. Nuclear runoff and mRNA stability assays will first establish if the lower levels reflect decreased transcription rates and/or mRNA stability. Present results suggest mRNA stability is not affected. Detailed mechanisms of these actions will be investigated by mutational and footprint analysis of the regulatory regions of the IL-2 gene.

拟议研究的广泛目标是了解在 分子和细胞水平糖皮质激素的生理作用， 特别是免疫系统细胞上的那些。这些行动说明， 糖皮质激素在临床上广泛应用， 免疫抑制剂和治疗淋巴细胞白血病， 淋巴瘤将进行研究以阐明（1）结构， 糖皮质激素各种磷酸化状态的产生和功能 受体;（2）糖皮质激素抑制的机制， 淋巴因子产生 (1)如PI所示。实验室用WEHI-7小鼠胸腺瘤细胞， 每个受体类固醇结合蛋白的磷酸盐平均数约为 3无激素，增加到约5与激素，并减少到3 或更少的盐提取的核部分。“受体”，形成 在ATP耗尽的细胞中，失去一个磷酸盐。部分蛋白水解揭示了 磷酸丝氨酸在类固醇结合和N-末端结构域，但不是在 DNA结合域。具体目标是：（a）通过研究确定 磷酸化的动力学，受体种类， 磷酸化底物。初步结果表明， 酶诱导的磷酸化是活化的受体。(b)定位 通过HPLC获得的磷酸肽部分测序的磷酸氨基酸 胰蛋白酶限度分析。初步HPLC分析， 未配体的受体揭示了两个主要的磷酸肽。(c)分析 磷酸化对生物活性影响 诱变和激酶和磷酸酶抑制剂。(d)识别激酶 磷酸化受体，使用激酶缺陷细胞，激酶和 磷酸酶抑制剂和合成肽底物。目前为止的证据 表明A激酶不参与。 (2)糖皮质激素免疫抑制可能主要是由于抑制 细胞因子的产生，其中白细胞介素-2（IL-2）和γ-干扰素 与mRNA水平降低有关。核径流和mRNA稳定性 如果较低的水平反映了 转录速率和/或mRNA稳定性。目前的结果表明， 稳定性不受影响。这些行动的详细机制将 通过调控区的突变和足迹分析进行研究 IL-2基因。