INSULIN STIMULATION OF PROLACTIN GENE EXPRESSION

描述（改编自申请人摘要）：胰岛素治疗 增加内源性催乳素基因的转录， 表达由来自大肠杆菌的5 ′-侧翼DNA组成的嵌合质粒， 催乳素基因连接到细菌氯霉素乙酰基 转移酶（CAT）基因在GH细胞。因此，该细胞培养系统 准确反映催乳素的生理调节， 胰岛素由于糖尿病引起的催乳素表达缺陷已经被发现， 建议解释几种疾病，包括婴儿呼吸道疾病， 抑郁症和阳痿这项研究的长期目标是 是发现这个规则的所有组成部分，以及它们是如何 相互作用引起催乳素基因表达增加。迄今为止的结果 已经确定了一个共识胰岛素反应元件（IRE）。这个IRE是 Ets基序相关序列CGGAA，它介导了100%的 胰岛素导致催乳素-CAT表达增加> 10倍。的 胸苷激酶和生长抑素中相同元件的缺失 启动子使它们对胰岛素不敏感，并确定CGGAA 共识IRE。胰岛素信号的传导需要 胰岛素受体具有完整的激酶功能，并依赖于 胰岛素受体的酪氨酸自磷酸化。 继续这些非常成功的研究的实验包括： 1)确认GABP介导100%的胰岛素反应， 击倒对手。2)GABP的MAP激酶磷酸化 α可能介导对胰岛素的反应，将进一步探讨。第三章 介导细胞凋亡所需的GABP α和GABP β突变的结构域 将确定对胰岛素的反应。GABP α和GABP β 突变将用于确定这些突变体是否介导胰岛素 对催乳素基因表达的影响。GABP α的嵌合蛋白- Gal4将与UAS-CAT报告器一起用于研究功能 GABP α的结构域。GABP-null细胞也将通过同源的 重组和突变体将在GABP-null环境中进行测试。 4)描述了几种用于鉴定和克隆 GABP可能与其他因子相互作用调节催乳素基因 表情

DESCRIPTION (Adapted from the applicant's abstract): Insulin treatment increases the transcription of the endogenous prolactin gene and the expression of chimeric plasmids consisting of 5'-flanking DNA from the prolactin gene ligated to the bacterial chloramphenicol acetyl transferase (CAT) gene in GH cells. Thus, this cell culture system accurately reflects the physiological regulation of prolactin by insulin. Defects in prolactin expression due to diabetes have been suggested to account for several disorders including infant respiratory distress syndrome and impotence. The long term goal of this research is to discover all of the components of this regulation and how they interact to cause increased prolactin gene expression. Results to date have identified a consensus insulin response element (IRE). This IRE is the Ets-motif-related sequence CGGAA and it mediates 100 percent of the >10-fold increase in prolactin-CAT expression caused by insulin. The deletion of identical elements in the thymidine kinase and somatostatin promoters renders them insensitive to insulin and establishes that CGGAA is consensus IRE. The transduction of the insulin signal requires insulin receptor with intact kinase function and is dependent on the tyrosine autophosphorylation of the insulin receptor. Experiments to continue these highly successful investigations include: 1) Confirm that GABP mediates 100 percent of the insulin response with knockouts. 2) Evidence that the MAP kinase phosphorylation of GABP alpha may mediate the response to insulin will be further explored. 3) Domains of GABP alpha and GABP beta mutations required to mediate the response to insulin will be determined. GABP alpha and GABP beta mutations will be used to determine if these mutants mediate insulin effects on prolactin gene expression. Chimeric proteins of GABP alpha- Gal4 will be used with an UAS-CAT reporter to investigate functional domains of GABP alpha. GABP-null cells will also be made by homologous recombination and mutants will be tested in a GABP-null environment. 4) Several approaches are described for identifying and cloning the other factors with which GABP might interact to regulate prolactin gene expression.