MECHANISM OF PROLACTIN ACTION

催乳素作用机制

• 批准号: 3315060
• 负责人: PAUL A KELLY
• 金额: $ 8.12万
• 依托单位: MCGILL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-08-01 至 1986-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3315060
• 关键词: electrofocusing; gel filtration chromatography; gene expression; high performance liquid chromatography; hormone regulation /control mechanism; ion exchange chromatography; messenger RNA; prolactin; radiotracer

I propose to study the mechanism of prolactin action. Prolactin's primary action involves the development of the mammary gland and lactation. This lactogenic hormone, like insulin and growth hormone, does not act via cyclic nucleotides. Recently we have been able to identify a soluble mediator of prolactin action which specifically stimulates casein messenger RNA production from isolated mammary nuclei. This mediator has been partially characterized. It is a small peptide of 1000-2000 daltons, heat stable and inactivated by trypsin. A new assay to measure casein gene expression, involving the insertion of a complementary cDNA probe to rabbit Alpha- or Beta-casein in the plasmid pBR322 will be used. We have been able to measure the specific hybridization of casein mRNA to the cloned cDNA on nitrocellulose filters. We are developing a "dot hybridization" assay for the rapid detection of multiple samples. This assay will be of great value in the purification of the soluble PRL mediator, which will be undertaken. Batches of rabbit mammary membranes will be exposed to PRL and the resulting soluble fraction will be lyophilized and passed over a Sephadex G-10 column. Classical purification procedures (ion exchange HPLC, gel exclusion, isoelectric focusing, etc.), will be utilized to purify this peptide. Following purification, the amino acid sequence will be determined and analogues will be developed. The mechanisms involved in the release of the mediator from membranes, the role of phosphatidylinositol metabolism, as well as the interaction of the mediator with DNA and the process of turning on the prolactin sensitive genes will be studied. The mechanism of action of other hormones, such as insulin, growth factors and gonadotropins will be compared to that of prolactin. It is possible that soluble mediators are released by a number of hormones, even perhaps by those which have been shown to act via cyclic nucleotides.

我建议研究催乳素的作用机制。 催乳素原发性 作用涉及乳腺的发育和泌乳。 这 催乳激素，如胰岛素和生长激素，不通过 环核苷酸 最近，我们已经能够确定一种可溶性的 一种能特异性刺激酪蛋白信使的催乳素作用介质 从分离的乳腺细胞核产生RNA。 这位调解人一直 部分特征。 它是一种1000-2000道尔顿的小肽， 稳定且被胰蛋白酶灭活。 一种检测酪蛋白基因的新方法 表达，包括插入互补cDNA探针到兔 将使用质粒pBR 322中的α-或β-酪蛋白。 我们一直 能够测量酪蛋白mRNA与克隆的 硝酸纤维素滤膜上的cDNA。 我们正在开发一种“斑点杂交” 用于快速检测多个样品的分析。 本试验将 在纯化可溶性PRL介体方面具有很大的价值，这将是 进行。 兔乳房膜批次将暴露于PRL， 将得到的可溶性级分冻干并通过 Sephadex G-10柱。 经典的纯化程序（离子交换 HPLC、凝胶排阻、等电聚焦等），将用于 纯化该肽。 纯化后，氨基酸序列将 并将开发类似物。 涉及的机制 从细胞膜释放介质， 磷脂酰肌醇代谢，以及介质的相互作用 启动催乳素敏感基因的过程 被研究。 其他激素的作用机制，如胰岛素， 将生长因子和促性腺激素与催乳素进行比较。 它 可溶性介质可能由许多激素释放， 甚至可能是那些已经显示通过环核苷酸起作用的化合物。