A novel intracellular 7TM estrogen receptor in breast

说明(申请人提供)：雌激素是人体内的一种重要荷尔蒙，调节许多不同的生理作用，包括许多组织的生长、发育和动态平衡。雌激素在正常乳腺组织的发育中起着关键作用，而对雌激素的反应性是乳腺癌患者最重要的预后和治疗因素之一。这些特性中的许多都归因于一种可溶的雌激素受体(ER)，它在很大程度上是作为配体激活的转录因子发挥作用的。我们已经鉴定了一种新的细胞内、7跨膜、G蛋白偶联的雌激素受体(GPR30)，我们提出了与ER一起调节细胞和组织对雌激素的反应性的功能。我们发现GPR30主要位于内质网中。目标1的目标是确定GPR30内调节其细胞位置的序列。我们还开发了一种新的荧光雌激素衍生物家族，可以与ER和GPR30结合。在目标2中，我们将使用这些荧光雌激素来开发GPR30的基于细胞的雌激素结合分析，并确定一系列雌激素相关化合物的结合特异性和亲和力。最近，我们在乳腺癌细胞中研究了GPRSO介导的新的信号转导通路。磷脂酰肌醇3，4，5-三磷酸的核积累是由雌激素介导的GPR30反式激活表皮生长因子受体(EGFR)启动的。目标3的目的是研究与EGFR磷酸化和内化有关的EGFR反式激活的机制，导致转录激活和细胞生长控制。最后，我们建立了一个GPR30缺失小鼠群体，对GPR30在乳房生长发育中的体内功能进行了研究。在目标4中，我们将鉴定GPR30在野生型小鼠中的表达，并分析GPR30缺失小鼠的表型。我们还将描述GPR30和经典ER都缺失的小鼠的表型。有了这些工具，我们可以在基因定义的背景下解决GPR30的雌激素反应信号。这些实验对于确定这种新的雌激素反应受体在雌激素介导的正常发育、肿瘤进展和乳腺癌治疗中的生长、增殖和分化中的作用将是非常有价值的。

DESCRIPTION (provided by applicant): Estrogen is a crucial hormone in the human body, regulating many diverse physiological effects, including the growth, development and homeostasis of numerous tissues. Estrogen plays a critical role in the development of normal breast tissue, whereas responsiveness to estrogen represents one of the most important prognostic and therapeutic factors for patients with breast cancer. Many of these properties have been attributed to a soluble receptor for estrogen (ER) that functions in large part as a ligand-activated transcription factor. We have characterized a novel intracellular, 7-transmembrane spanning, G protein- coupled estrogen receptor (GPR30) that we propose functions alongside ER to regulate cell and tissue responsiveness to estrogen. We have discovered that GPR30 resides predominantly in the endoplasmic reticulum. The goal of Aim 1 is to determine the sequences within GPR30 that regulate its cellular location. We have also developed a novel family of fluorescent estrogen derivatives that binds to both the ER and GPR30. In Aim 2 we will use these fluorescent estrogens to develop cell-based estrogen binding assays for GPR30 and determine the binding specificities and affinities of a collection of estrogen-related compounds. Recently, we have characterized novel GPRSO-mediated signal transduction pathways in breast cancer cells. The nuclear accumulation of phosphatidylinositol 3,4,5-trisphosphate is initiated by estrogen-mediated transactivation of epidermal growth factor receptor (EGFR) by GPR30. The goal of Aim 3 is to investigate the mechanisms involved in EGFR transactivation with respect to EGFR phosphorylation and internalization, leading to transcriptional activation and cell growth control. Finally, we have established a colony of GPR30 null mice to dissect the in vivo function of GPR30 in breast growth and development. In Aim 4, we will characterize GPR30 expression in wild type mice and analyze the phenotypes of GPR30 null mice. We will also characterize the phenotypes of mice in which both GPR30 and the classical ERs have been deleted. With these tools, we can address estrogen-responsive signaling of GPR30 in a genetically defined background. The experiments proposed will be invaluable in defining the roles of this novel estrogen- responsive receptor in estrogen-mediated growth, proliferation, and differentiation in normal development as well as in neoplastic progression and the treatment of breast cancer.