High Resolution Analysis of Integrated Subplasmalemmal Calcium and Oxidant Signaling Mechanisms in Gonadotropes

项目摘要/摘要 通常被称为生殖的“第一”荷尔蒙，下丘脑多肽促性腺激素释放激素 促性腺激素释放激素(GnRH)是激活下丘脑-垂体-性腺轴(HPG)的关键和必要的内分泌信号 因此，雄性和雌性的生殖功能也是如此。从正中隆起的轴突终末释放出来， 促性腺激素释放激素通过垂体门循环输送到脑下垂体前叶，在那里它与高密度脂蛋白结合。 促性腺激素释放激素受体与促性腺激素促黄体生成素的表达和释放 促黄体生成素(LH)和卵泡刺激素(FSH)。在女性中，促黄体生成素的急剧升高是诱导排卵的必要条件。 因此，对于生殖和生育来说，这是一个强制性的事件。在过去的几十年里，有很多事情 阐明促性腺激素释放激素受体激活所引发的关键细胞和生化事件 包括促性腺激素基因变化背后的细胞内信号中间产物的身份 表达和分泌。然而，缺少的是技术的开发和应用，这些技术 具有分析GnRH引发的即时早期事件所需的空间和时间分辨率 在活细胞中。这类事件包括质膜信号域的形成。最近，我们 已经应用高分辨率成像来演示GnRH介导的活性氧物种的产生 (ROS)再加上L型钙通道的开放。钙和活性氧物种(ROS)是 无处不在的信号分子，影响细胞过程，从神经递质释放到 细胞凋亡。这项提案的总体目标是调查知之甚少的控制机制 垂体促性腺激素中的钙和ROS信号机制。更具体地说，这项研究调查了 氧化剂和钙信号微域功能定位的一种新的调控机制 在促性腺激素释放激素受体刺激后汇聚成促性腺激素。这促进了RO的增加 而L型钙通道的生成和活动，增加了细胞内的钙离子，最终改变了细胞内的钙离子。 排卵所需的基因表达。 在这个应用中，我们建议测试一个模型，其中钙和氧化剂微域的聚合 信号转导与ERK信号的激活有关。我们还将调查潜在的机制 调节这一信号模式。特异性目标1检验亚质膜促性腺激素释放激素诱导的假设 钙和ROS微区共定位，并在功能上偶联。《特定目标2》检验了这一假设 激活的GnRH受体组装并形成动态的多蛋白信号复合体 共定位的钙和ROS微域转导到ERK激活的特异性目标3测试 肌动蛋白细胞骨架动力学受氧化调控并对GnRH受体至关重要的假说 微域信令。在这些特定目标的实验中将使用电压钳位组合 电生理学、全内反射荧光显微镜、分子生物学、生物化学 超分辨率成像和蛋白质组学来检查这种局部信号的结构和功能 机制。这些实验的结果将提供对潜在事件的机械性洞察 促性腺激素功能和功能障碍，并可能导致开发新的合理的治疗方法 调节GnRH受体信号与生育和其他重要的健康问题，如GnRH 受体反应性癌症。

Project Summary/Abstract Often called the “first” hormone of reproduction, the hypothalamic peptide gonadotropin releasing hormone (GnRH) is the key and essential endocrine signal that activates the hypothalamic-pituitary-gonadal (HPG) axis and, thus, reproductive function in males and females. Released from axon terminals in the median eminence, GnRH is transported via the hypophyseal portal circulation to the anterior pituitary gland where it binds to high affinity GnRH receptors and stimulates the expression and release of the gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH). In females, an acute rise in LH is obligatory for inducing ovulation and as such is a mandatory event for reproduction and fertility. Over the last several decades much has been elucidated regarding the key cellular and biochemical events elicited by activation of the GnRH receptor including the identity of intracellular signaling intermediates that underlie changes in gonadotropin gene expression and secretion. Missing, however, has been the development and application of technologies that possess the spatial and temporal resolution necessary for analyzing immediate early events elicited by GnRH in living cells. Such events would include the formation of plasma membrane signaling domains. Recently, we have applied high resolution imaging to demonstrate GnRH-mediated production of reactive oxygen species (ROS) coupled to the opening of L-type calcium channels. Calcium and reactive oxygen species (ROS) are ubiquitous signaling molecules that influence cellular processes ranging from neurotransmitter release to apoptosis. The general goal of this proposal is to investigate the poorly understood mechanisms controlling calcium and ROS signaling mechanisms in pituitary gonadotropes. More specifically, this research investigates a novel regulatory mechanism where localized oxidant and calcium signaling microdomains functionally converge in gonadotropes following stimulation of the GnRH receptor. This promotes increased ROS generation and L-type calcium channel activity, increased calcium within the cells, and ultimately changes in gene expression required for ovulation. In this application we propose to test a model where the convergence of calcium and oxidant microdomain signaling is coupled to activation of ERK signaling. We will also investigate the underlying mechanisms regulating this signaling modality. Specific Aim 1 tests the hypothesis that subplasmalemmal GnRH-induced calcium and ROS microdomains colocalize and are functionally coupled. Specific Aim 2 tests the hypothesis that activated GnRH receptors assemble and form dynamic multi-protein signaling complexes necessary for transduction of colocalized calcium and ROS microdomains to ERK activation Specific Aim 3 tests the hypothesis that actin cytoskeletal dynamics are regulated oxidatively and are essential for GnRH receptor microdomain signaling. The experiments in these Specific Aims will use a combination of voltage-clamp electrophysiology, total internal reflection fluorescence (TIRF) microscopy, molecular biology, biochemistry, super resolution imaging, and proteomics to examine the structural and function of this local signaling mechanism. The outcome of these experiments will provide mechanistic insights into events underlying gonadotrope function and dysfunction and may lead to the development of new rational approaches for regulating GnRH receptor signaling with respect to fertility and other important health issues such as GnRH receptor-responsive carcinomas.