REGULATION OF GNRH RECEPTOR GENE EXPRESSION

促性腺激素释放激素(GnRH)是由 下丘脑的神经内分泌细胞。在绑定到特定 受体位于垂体前叶促性腺激素上，GnRH不仅 刺激而非S合成分泌的黄体生成素和， 在较小程度上，FSH。鉴于促性腺激素释放激素在 控制哺乳动物的生殖功能已经投入了大量的努力 有助于理解调节这一过程的生理后果 激素及其同源受体。例如，哺乳动物的排卵是 依赖于黄体生成素分泌速率的短暂增加，在 反过来，结果是GnRH分泌增加和GnRH增加 垂体促性腺激素释放激素受体的浓度。最近可获得的基因片段 编码促性腺激素释放激素受体的克隆可以直接测量mRNA 和研究人员开始确定GnRH受体的 转录机制在调节变化中的生理学意义 促性腺激素释放激素受体的数量。然而，由于缺乏基因组克隆， GnRH受体排除了对潜在分子机制的任何分析 促性腺激素释放激素受体基因转录调控。在此，我们报告 小鼠促性腺激素释放激素受体部分基因组克隆的分离 位于He 5‘端的细胞特异性启动子的初步鉴定 侧翼区域。因此，我们准备开始对 顺式作用的DNA元件和反式作用因子负责细胞- GnRH受体基因的特异性和激素介导的表达。我们的 长期目标是确定两者背后的分子机制 促性腺激素释放激素受体的组织特异性表达 吉恩。因此，在具体目标1中，我们将使用瞬变表达式 AlphaT3细胞中的分析、DNA-蛋白质结合分析和脂质体介导 基因转移研究对细胞特异性表达的要求 小鼠GnRH受体基因。在具体目标2中，我们将使用站点定向 突变和瞬时表达分析以确定该基因的区域 小鼠GnRH受体基因启动子对GnRH、PMA的反应性 或者露营。此外，我们还将确定小鼠GnRH受体基因是否包含 雌激素受体高亲和力结合部位(S)。在具体目标3中，我们 建议构建转基因小鼠作为细胞特异性的终极测试 促性腺激素释放激素受体嵌合基因的表达及其作为体内模型的研究 用于研究激素对促性腺激素释放激素受体基因表达的调节。 最后，在确定顺位作用因素方面取得的进展有限。 促性腺激素基因GnRH激活所需的反式作用因子 表情。因此，我们提出了第四个具体目标，以解决 促性腺激素释放激素刺激血管内皮生长因子表达的分子要求 糖蛋白激素α亚单位基因。

Gonadotropin-releasing hormone (GnRH) is synthesized and secreted from neuroendocrine cells of the hypothalamus. Upon binding to specific receptors located on gonadotropes of the anterior pituitary, GnRH not only stimulates but is obligatory for the s synthesis and secretion of LH and, to a lesser extent, FSH. In light of the pivotal role of GnRH in controlling reproductive function of mammals much effort has been devoted toward understanding the physiological consequences of regulation of this hormone and its's cognate receptor. For example, ovulation in mammals is dependent on a transitory increase in the secretory rate of LH that, in turn, results from both an increase in GnRH secretion and an increase in pituitary concentration of GnRH receptors. Recent availability of cDNA clones encoding the GnRH receptor has permitted direct measurement of mRNA for the GnRH receptor and researchers are beginning to establish the physiological importance of transcriptional mechanisms in mediating changes in the number of GnRH receptors. However, lack of genomic clones for the GnRH receptor has precluded any analysis of molecular mechanisms underlying transcriptional regulation of the GnRH receptor gene. Herein, we report the isolation of a partial genomic clone for the murine GnRH receptor and preliminary characterization of a cell-specific promoter located int he 5' flanking region. Thus, we are poised to begin a systematic analysis of the cis-acting DNA elements and trans-acting factors responsible for cell- specific and hormonally mediated expression of the GnRH receptor gene. Our long-term goals are to define the molecular mechanisms underlying both tissue-specific and hormonally-regulated expression of the GnRH receptor gene. Accordingly, in Specific Aim 1, we will use transient expression assays in alphaT3 cells, DNA-protein binding assays, and liposome-mediated gene transfer to study the requirements for cell-specific expression of the mouse GnRH receptor gene. In Specific Aim 2, we will use site-directed mutagenesis and transient expression assays to identify regions of the mouse GnRH receptor gene promoter that confer responsiveness to GnRH, PMA or cAMP. Also, we will determine if the mouse GnRH receptor gene contains high affinity binding site(s) for estrogen receptor. In Specific Aim 3, we propose to construct transgenic mice as an ultimate test for cell-specific expression of chimeric GnRH receptor genes and to serve as an in vivo model for studying hormonal regulation of GnRH receptor gene expression. Finally, limited progress has been made in identifying cis-acting elements or trans-acting factors required for GnRH activation of gonadotropin gene expression. Thus, we propose a fourth specific aim to address the molecular requirements for GnRH stimulation of expression of the glycoprotein hormone alpha subunit gene.