MODE OF ACTION OF STEROID HORMONES

类固醇激素的作用方式

• 批准号: 3224391
• 负责人: ALLAN U MUNCK
• 金额: $ 33.99万
• 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/3224391
• 关键词: DNA footprinting; antiinflammatory agents; enzyme substrate; genetic transcription; glucocorticoids; high performance liquid chromatography; hormone regulation /control mechanism; immunosuppression; interferons; 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.

拟议研究的主要目标是了解以下内容 分子和细胞水平糖皮质激素的生理作用， 尤其是免疫系统细胞上的那些。这些行动说明了 糖皮质激素作为抗炎和抗肿瘤药物的广泛临床应用 免疫抑制剂和在治疗淋巴细胞白血病和 淋巴瘤。将进行研究以阐明(1)结构， 糖皮质激素不同磷酸化状态的产生和功能 细胞中的受体；(2)糖皮质激素抑制血管紧张素转换酶的机制 淋巴因子制作。 (1)如P.I.‘S实验室用WEHI-7小鼠胸腺瘤细胞所显示的那样， 每个受体类固醇结合蛋白的平均磷酸盐数量约为 3在没有激素的情况下，有激素时增加到5左右，然后减少到3 或更少在可抽提的核分数中。形成的“零受体” 在ATP耗尽的细胞中，会失去一种磷酸盐。部分蛋白分解揭示了 类固醇结合区和N-端区中的磷酸丝氨酸，而不是 DNA结合域。具体目标是：(A)通过研究确定 关于磷酸化的动力学，受体种类是 用于磷酸化的底物。初步结果表明，底物 激素诱导的磷酸化是激活的受体。(B)找到 用高效液相色谱法测定磷酸多肽的部分序列 限制性胰酶消化的分析。高效液相色谱仪的初步分析 未连接的受体揭示了两个主要的磷酸肽。(C)分析 利用定点定位技术研究磷酸化对生物活性的影响 诱变与激酶和磷酸酶抑制剂。(D)确定激活酶 使受体磷酸化，使用缺乏激酶的细胞、激酶和 磷酸酶抑制剂和合成肽底物。到目前为止的证据 表明A-激酶没有参与。 (2)糖皮质激素免疫抑制可能主要是由于抑制 白介素2(IL-2)和γ-干扰素的细胞因子产生 与信使核糖核酸水平降低有关。核径流与信使核糖核酸稳定性 化验将首先确定较低的水平是否反映了下降 转录速率和/或信使核糖核酸稳定性。目前的研究结果表明， 稳定性不受影响。这些行动的详细机制如下 通过对调节区的突变和足迹分析进行研究 白介素2基因。