REGULATION OF GNRH RECEPTOR GENE EXPRESSION

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是生殖的核心。在缺乏GnRH输入的情况下，促黄体生成素的合成和分泌，因此，正常的性腺功能停止。因此，GnRH受体（GnRHR）是接受和介导促性腺激素主要刺激输入的部位。我们发现在促性腺激素来源的alpha -1细胞中，小鼠GnRHR的表达是由一个复杂的增强子介导的，该增强子的成分包括类固醇生成因子-1 （SF- 1）的结合位点、一个AP-1元件和一个我们称之为GnRH受体激活序列（GRAS）的元件。这种复杂的增强剂还集成了多种内分泌输入。首先，我们最近发现GRAS与GnRHR启动子的激活调节共定位。然而，尚未解决的是在GRAS中整合功能活性的蛋白的身份。在Specific Aim 1中，我们建议鉴定调节GRAS功能活性和激活蛋白反应的蛋白。其次，AP-1似乎是介导GnRH调节的操作元件；然而，关于最终导致GnRH在GnRHR AP-1位点激活的信号转导级联和下游靶标的重要问题仍然存在。在Specific Aim 2中，我们的目标是明确GnRH调控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反应性。在Specific Aim 3中，我们建议扩展这些研究，进一步探索GnRHR基因在组织/细胞特异性表达和激素调控方面的要求。最后，我们已经产生了表达GnRHR本质荧光形式的细胞系。这些分子提供了一个独特的机会来研究GnRHR作为活细胞中被占用和未被占用的受体。在Specific Aim 4中，我们将使用荧光共振能量转移来验证GnRH信号传导的早期事件是激动剂诱导的受体自我关联的假设。在生育调节方面，研究GnRH及其同源受体的相关性是明确的。然而，在肌瘤、子宫内膜异位症、乳腺癌、前列腺癌、睾丸癌和垂体癌的治疗中，GnRH的强效激动剂和拮抗剂的使用强调了对GnRH和GnRHR在健康和疾病中的充分理解的必要性。

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.