INSULIN STIMULATION OF PROLACTIN GENE EXPRESSION

描述(改编自申请人的摘要)：胰岛素治疗 增加内源性催乳素基因的转录和 沙门氏菌5‘-侧翼DNA嵌合载体的表达 催乳素基因与细菌氯霉素乙酰化的连接 生长激素细胞中的转移酶(CAT)基因。因此，这个细胞培养系统 准确地反映了催乳素的生理调节 胰岛素。由于糖尿病导致的催乳素表达缺陷 建议解释几种疾病，包括婴儿呼吸道疾病 窘迫综合征和阳萎。这项研究的长期目标是 是发现这一法规的所有组成部分，以及它们如何 相互作用导致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的胰岛素反应元件(IRE)。这场愤怒是 Ets-Motif相关序列CGGAA和它100%介导 胰岛素使催乳素-CAT的表达增加10倍。这个 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，并证实CGGAA 是一致的愤怒。胰岛素信号的传递需要 具有完整的激酶功能的胰岛素受体，并依赖于 胰岛素受体的酪氨酸自动磷酸化。 继续这些非常成功的调查的实验包括： 1)确认GABP 100%介导胰岛素反应 淘汰赛。2)有证据表明GABP的MAP激酶磷酸化 阿尔法可能对胰岛素的反应起调节作用还有待进一步探讨。3) GABPα和GABPβ突变所需的结构域 对胰岛素的反应将被确定。GABPα和GABPβ 突变将被用来确定这些突变是否介导了胰岛素 对催乳素基因表达的影响。GABPα-嵌合蛋白 Gal4将与UAS-CAT记者一起使用，以调查功能 GABPα的结构域。GABP阴性细胞也将由同源 重组和突变体将在GABP为零的环境中进行测试。 4)描述了识别和克隆的几种方法 GABP可能与其相互作用调节催乳素基因的其他因素 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.