REGULATION OF GNRH RECEPTOR GENE EXPRESSION

促性腺激素释放激素(GnRH)与位于脑下垂体前叶促性腺激素细胞上的高亲和力受体结合是生殖的中心。在没有GnRH输入的情况下，黄体生成素的合成和分泌，从而停止正常的性腺功能。因此，GnRH受体(GnRHR)是接收和介导对促性腺激素的初级刺激输入的部位。我们发现小鼠GnRHR在促性腺激素来源的αT3-1细胞中的表达是由一个复合增强子介导的，其成分包括类固醇生成因子-1(SF-1)的结合位点、AP-1元件和我们称为GnRH受体激活序列(GRAS)的元件。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是共定位的。然而，尚未解决的是整合GRAS功能活动的蛋白质(S)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(S)。第二，AP-1似乎是介导GnRH调节的操作元件；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，仍然存在重要的问题。在特定的目标2中，我们的目标是确定GnRH调节GnRHR基因表达的分子机制。我们还发现，1900bp的近端启动子足以在转基因小鼠中进行组织特异性表达和GnRH反应。在具体目标3中，我们建议扩大这些研究，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，我们已经产生了表达GnRHR固有荧光形式的细胞系。这些分子为研究GnRHR在活细胞中既被占据又未被占据的受体提供了独特的机会。在特定的目标4中，我们将使用荧光共振能量转移来验证GnRH信号中的早期事件是激动剂诱导的受体自结合的假设。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，GnRH的强效激动剂和拮抗剂在治疗子宫肌瘤、子宫内膜异位症以及乳腺癌、前列腺癌、睾丸和脑垂体癌方面的使用强调了在健康和疾病中充分了解GnRH和GnRHR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.