Membrane Events Following Hormone Binding to LH Receptor

说明：(申请人提供)功能性黄体生成素 受体对男性和女性的生育能力都至关重要。这些受体 也很重要，因为越来越多的证据表明自然发生的， 黄体生成素受体的结构性活性突变导致性早熟 并与卵巢和睾丸的发育有关 肿瘤。这项研究的总体目标是了解 促黄体生成素受体结构在男女调节中的作用 生育能力。在目标I中，我们将确定分离的促黄体生成素受体是否形成二聚体 或激素结合后的低聚物。时间分辨磷光 黄体生成素受体旋转扩散和荧光的各向异性测量 将使用单一受体的成像。在目标2中，我们将探讨 促黄体生成素受体在信号传递中的自关联。我们将确定是否 固有活性的促黄体生成素受体是自相关的，是否广泛 黄体生成素受体的交联会产生信号。在目标3中，我们会问 黄体生成素受体在激素结合后进入脂筏，以及 受体的自我结合必然先于这种易位。筏 活细胞上单个黄体生成素受体的分离与示踪 将被用来确定促黄体生成素受体是否在小鼠 激素结合后的膜微区。在目标4中，我们将确定 黄体生成素受体脱敏是否发生在膜筏内。这种隔绝 在受体脱敏后不同时间的膜筏 确定是否存在黄体生成素受体有计划地进出 木筏。我们实验室研制了用于细胞的激光光学仪器 几乎没有其他实验室能与之匹敌的膜研究。技术的应用 这些工具方法将提供对膜事件的洞察 伴随的促黄体生成素受体介导的信号转导和受体 脱敏。这些研究也将借鉴国际和平研究所的经验。 以及科林·克莱博士的专业知识，他是动物繁殖部的成员 和生物技术实验室，以及神经生物学家迈克尔·塔姆昆博士 科罗拉多州立大学。

DESCRIPTION: (provided by applicant) Functional luteinizing hormone (LH) receptors are critical to fertility in both males and females. These receptors are also important because of increasing evidence that naturally-occurring, constitutively active mutations of the LH receptor lead to precocious puberty in males and are implicated in the development of ovarian and testicular tumors. The overall goal of this research is to understand the role of luteinizing hormone receptor structure in regulation of male and female fertility. In Aim I we will determine whether isolated LH receptors form dimers or oligomers following hormone binding. Time-resolved phosphorescence anisotropy measurements of LH receptor rotational diffusion and fluorescence imaging of single receptors will be used. In Aim 2 we will explore the role of the LH receptor self-association in signaling. We will determine whether constitutively active LH receptors are self-associated and whether extensive crosslinking of LH receptors results in signaling. In Aim 3 we will ask whether LH receptors move into lipid rafts following binding of hormone and whether receptor self-association necessarily precedes this translocation. Raft isolation together with tracking of individual LH receptors on viable cells will be used to determine whether LH receptors become localized in small membrane microdomains following binding of hormone. In Aim 4 we will determine whether LH receptor desensitization occurs within membrane rafts. The isolation of membrane rafts at various times following receptor desensitization will establish whether there is orchestrated movement of LH receptors into and out of rafts. Our laboratory has developed laser optical instrumentation for cell membrane studies that are equaled by few other laboratories. The application of these instrumental methods will provide insight into membrane events accompanying LH-receptor mediated signal transduction and receptor desensitization. These studies will also draw on the experience of the PI as well as the expertise of Dr. Colin Clay, a member of the Animal Reproduction and Biotechnology Laboratory, and of Dr. Michael Tamkun, a neurobiologist at Colorado State University.