REGULATION OF GNRH RECEPTOR GENE EXPRESSION

促性腺激素释放激素（GnRH）是合成和分泌的， 下丘脑的神经内分泌细胞。 在结合到特定的 受体位于促性腺激素的垂体前叶，GnRH不仅 刺激LH的合成和分泌， 在某种程度上，FSH。 鉴于促性腺激素释放激素在 为了控制哺乳动物的生殖功能， 为了理解这种调节的生理后果， 激素及其同源受体。 例如，哺乳动物的排卵是 依赖于LH分泌率的短暂增加， 反过来，这是由于GnRH分泌增加和 垂体GnRH受体浓度。 cDNA的最新可用性 编码GnRH受体的克隆允许直接测量mRNA 研究人员开始建立一种 转录机制在介导变化中的生理重要性 GnRH受体的数量。 然而，缺乏基因组克隆， GnRH受体排除了任何潜在的分子机制分析 促性腺激素释放激素受体基因的转录调控。 在此我们报告 分离鼠GnRH受体的部分基因组克隆， 位于5 '端的细胞特异性启动子的初步鉴定 侧翼区 因此，我们准备开始系统地分析 顺式作用DNA元件和反式作用因子， GnRH受体基因的特异性和生殖介导的表达。 我们 长期目标是确定两者的分子机制 GnRH受体的组织特异性和生殖调控表达 基因 因此，在具体目标1中，我们将使用瞬时表达 α T3细胞中的测定、DNA-蛋白质结合测定和脂质体介导的 基因转移研究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 突变和瞬时表达测定以鉴定 小鼠促性腺激素释放激素受体基因启动子，赋予对促性腺激素释放激素，PMA的反应性 或cAMP。 此外，我们将确定小鼠GnRH受体基因是否含有 雌激素受体的高亲和力结合位点。 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最终测试， 嵌合GnRH受体基因的表达并用作体内模型 用于研究GnRH受体基因表达的激素调节。 最后，在确定顺式作用元件方面取得的进展有限 或促性腺激素释放激素基因激活所需的反式作用因子 表情 因此，我们提出第四个具体目标， 促性腺激素释放激素刺激表达的分子要求 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.