REGULATION OF GNRH RECEPTOR GENE EXPRESSION

GNRH受体基因表达的调控

• 批准号: 6627377
• 负责人: Colin M Clay
• 金额: $ 37.83万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6627377
• 关键词: biological signal transduction; cell line; gene expression; genetic promoter element; genetically modified animals; gonadotropin releasing factor; hormone receptor; hormone regulation /control mechanism; laboratory mouse; protein structure function; tissue /cell culture

The binding of gonadotropin-releasing hormone (GnRH) to specific, high-affinity receptors located on gonadotrope cells of the anterior pituitary gland is central to reproduction. In the absence of GnRH input, synthesis and secretion of luteinzing hormone and, consequently, normal gonadal function ceases. Thus, the GnRH receptor (GnRHR) is the site that receives and mediates the primary stimulatory input to gonadotropes. We have found that expression of the murine GnRHR in gonadotrope-derived alphaT3-1 cells is mediated by a complex enhancer whose components include a binding site for steroidogenic factor-1 (SF- 1), an AP-1 element, and an element we have termed the GnRH receptor activating sequence (GRAS). This complex enhancer also integrates multiple endocrine inputs. First, we have recently found that GRAS co-localizes with activin regulation of the GnRHR promoter. Unresolved, however, is the identity of the protein(s) that integrate functional activity at GRAS. In Specific Aim 1, we propose to identify the protein(s) that regulate the functional activity, and activin responsiveness of GRAS. Second, AP-1 appears to be the operative element that mediates GnRH regulation; however, important questions remain as to the signal transduction cascades and downstream targets that ultimately lead to GnRH activation at the GnRHR AP-1 site. In Specific Aim 2, our goal is to define the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. We have also found that 1900 bp of proximal promoter is sufficient for tissue-specific expression and GnRH responsiveness in transgenic mice. In Specific Aim 3, we propose to expand these studies to further explore the requirements for tissue/cell-specific expression and hormonal regulation of the GnRHR gene. Finally, we have generated cell lines that express intrinsically fluorescent forms of the GnRHR. These molecules provide a unique opportunity to study the GnRHR as both an occupied and unoccupied receptor in living cells. In Specific Aim 4, we will use fluorescence resonance energy transfer to test the hypothesis that an early event in GnRH signaling is agonist induced receptor self- association. In terms of fertility regulation, the relevance of investigating GnRH and its cognate receptor is clear. However, the use of potent agonists and antagonists of GnRH in the treatment of fibroid tumors, endometriosis, and carcinomas of the breast, prostate, testes, and pituitary underscores the need for a full understanding of GnRH and the GnRHR in both health and disease.

促性腺激素释放激素 (GnRH) 与位于垂体前叶促性腺细胞上的特异性高亲和力受体的结合对于生殖至关重要。 在缺乏 GnRH 输入的情况下，黄体生成素的合成和分泌以及正常的性腺功能都会停止。 因此，GnRH 受体 (GnRHR) 是接收和介导促性腺激素主要刺激输入的位点。 我们发现促性腺激素衍生的 αT3-1 细胞中鼠 GnRHR 的表达是由复杂的增强子介导的，其成分包括类固醇生成因子 1 (SF-1) 的结合位点、AP-1 元件和我们研究的元件。称为 GnRH 受体激活序列 (GRAS)。 这种复杂的增强剂还整合了多种内分泌输入。 首先，我们最近发现 GRAS 与 GnRHR 启动子的激活素调节共定位。 然而，尚未解决的是在 GRAS 上整合功能活性的蛋白质的身份。 在具体目标 1 中，我们建议鉴定调节 GRAS 功能活性和激活素反应性的蛋白质。 其次，AP-1 似乎是介导 GnRH 调节的有效元件；然而，关于最终导致 GnRHR AP-1 位点 GnRH 激活的信号转导级联和下游靶点仍然存在重要问题。 在具体目标 2 中，我们的目标是定义 GnRH 调节 GnRHR 基因表达的分子机制。 我们还发现，1900 bp 的近端启动子足以在转基因小鼠中实现组织特异性表达和 GnRH 反应。 在具体目标 3 中，我们建议扩展这些研究，以进一步探索 GnRHR 基因的组织/细胞特异性表达和激素调节的要求。 最后，我们生成了表达 GnRHR 内在荧光形式的细胞系。 这些分子提供了一个独特的机会来研究 GnRHR 作为活细胞中占据和未占据的受体。 在具体目标 4 中，我们将使用荧光共振能量转移来检验以下假设：GnRH 信号传导的早期事件是激动剂诱导的受体自关联。 在生育调节方面，研究 GnRH 及其同源受体的相关性是显而易见的。 然而，GnRH 的强效激动剂和拮抗剂在治疗纤维瘤、子宫内膜异位症以及乳腺癌、前列腺癌、睾丸癌和垂体癌中的使用强调了充分了解 GnRH 和 GnRHR 在健康和疾病中的必要性。