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

项目总结/摘要 下丘脑肽促性腺激素释放激素通常被称为生殖的“第一”激素 促性腺激素释放激素（GnRH）是激活下丘脑-垂体-性腺（HPG）轴的关键和必需的内分泌信号 因此也影响了雄性和雌性的生殖功能。从正中隆起的轴突终末释放， GnRH通过垂体门脉循环转运到垂体前叶，在那里它与高浓度的 亲合GnRH受体，刺激促性腺激素促黄体激素的表达和释放 (LH)和促卵泡激素（FSH）。在女性中，LH的急剧升高是诱导排卵的必要条件 因此是繁殖和生育的必要事件。在过去的几十年里， 阐明了GnRH受体激活引起的关键细胞和生化事件 包括促性腺激素基因改变的细胞内信号中间体的身份 表达和分泌。然而，缺少的是技术的开发和应用， 具有分析GnRH引起的即刻早期事件所需的空间和时间分辨率 在活细胞中。这些事件包括质膜信号传导结构域的形成。最近我们 已经应用高分辨率成像来证明GnRH介导的活性氧的产生 (ROS)与L型钙通道的开放相关联。钙和活性氧（ROS）是 无处不在的信号分子，影响细胞过程，从神经递质的释放， 凋亡这项建议的总体目标是调查知之甚少的机制控制 垂体促性腺激素细胞中的钙和活性氧信号机制。更具体地说，这项研究调查了 一种新的调节机制，其中局部氧化剂和钙信号微区功能 在促性腺激素释放激素受体的刺激后会聚在促性腺激素细胞中。这促进了ROS的增加 产生和L型钙通道活性，增加细胞内的钙，并最终改变 排卵所需的基因表达。 在这个应用中，我们提出测试一个模型，其中钙和氧化剂微域的收敛 信号传导与ERK信号传导的激活偶联。我们还将研究潜在的机制， 调节这种信号传导方式。特异性目的1检验了质膜下GnRH诱导的 钙和ROS微区共定位并在功能上偶联。具体目标2检验假设 激活的GnRH受体组装并形成动态的多蛋白信号复合物， 将共定位的钙和ROS微区转导至ERK活化特异性Aim 3测试了 肌动蛋白细胞骨架动力学受氧化调节并对GnRH受体至关重要假说 微区信号这些特定目标中的实验将使用电压钳和电压钳的组合。 电生理学、全内反射荧光（TIRF）显微术、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传导的结构和功能 机制这些实验的结果将提供对事件的机械见解， 促性腺激素功能和功能障碍，并可能导致新的合理方法的发展， 调节GnRH受体信号传导与生育和其他重要的健康问题，如GnRH 受体反应性癌

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.