Extracellular ATP Metabolism as a Novel Regulator of Gonadotrope Cell Function

 描述(申请人提供)：促性腺激素释放激素(GnRH)是控制促性腺激素的生物合成和分泌的中枢神经内分泌介质。促性腺激素释放激素受体(R)作为下丘脑信号的中枢整合因子，在体内协调促性腺激素的行为。如果缺少这一关键的神经内分泌界面，生殖就会停止。GnRHR是一种独特的G蛋白偶联受体，在RAFT的隔室中显示结构性的质膜定位；这种膜的隔化是有效的细胞信号转导途径所必需的，在 雌性哺乳动物。在这个修订的应用中，我们将注意力集中在新的鉴定上，即F0F1 ATP合成酶复合体和电子传递链中的酶与GnRHR在垂体促性腺激素的膜筏中共存。在这个探索性的R21应用中，独特的初步蛋白质组学研究已经确定了一套129个与GnRHR特异定位在质膜上的多肽，这为探索这些膜相关复合体如何控制GnRH的作用和潜在的生育提供了一个强有力的机会。支持该研究计划的初步研究侧重于在促性腺激素细胞模型系统中，通过与GnRHR和膜筏标记Flotillin 1的相互作用，在细胞表面确定的F0F1 ATP合成酶复合体的功能重要性。这些研究表明，促性腺激素可以主动合成和代谢细胞外的ATP。此外，细胞外ATP张力调节GnRH在外植体培养中诱导的小鼠脑垂体促黄体生成素的分泌。在这些初步研究的基础上，我们的中心工作假设是，GnRHR在膜筏内的激活是由细胞外ATP通过F0F1 ATP合成酶和胞外NTPDase CD39作为细胞表面旁分泌效应器来调节的。ATP旁分泌对GnRH的调节作用会影响黄体生成素的分泌。目的1.分析电子传输链在调节促性腺激素细胞表面F0F1-ATP合成酶活性中的作用。目的2.确定胞外NTPDase CD39在调节促性腺激素释放激素作用中的生理意义。目的3.明确F0F1三磷酸腺苷合成酶代谢产物在细胞外空间的来源。

 DESCRIPTION (provided by applicant): Gonadotropin releasing hormone (GnRH) is the central neuroendocrine mediator controlling the biosynthesis and secretion of gonadotropic hormones. The GnRH receptor (R) serves as the central integrator of hypothalamic signals to coordinate gonadotrope behavior in vivo. In the absence of this critical neuroendocrine interface, reproduction ceases. The GnRHR is a unique G-protein-coupled receptor that displays constitutive plasma membrane localization in raft compartments; this membrane compartmentalization is required for efficient cell signaling to pathways critical for fertility in female mammals. In this revised application, we focus our attention on the novel identification of co-localization of the F0F1 ATP synthase complex and enzymes in the electron transport chain with the GnRHR within membrane rafts in pituitary gonadotropes. Unique preliminary proteomic studies in this exploratory R21 application have identified a suite of 129 peptides that specifically localize with the GnRHR at the plasma membrane providing a vigorous opportunity to explore how these membrane-associated complexes control GnRH action and potentially fertility. Preliminary studies in support of the proposed research plan focus on the functional importance of the F0F1 ATP synthase complex identified at the cell surface through interactions with the GnRHR and the membrane raft marker flotillin 1 in a gonadotrope cell model system. These studies demonstrate that the gonadotrope can actively synthesize and metabolize ATP extracellularly. Further, extracellular ATP tone modulates GnRH-induced secretion of LH from mouse pituitaries in explant culture. Based upon these preliminary studies, our central working hypothesis that activation of the GnRHR within membrane rafts is modulated by extracellular ATP through the actions of F0F1 ATP synthase and ectoNTPDase CD39 as paracrine effectors at the cell surface. ATP paracrine modulation of GnRH action will impact the secretion of LH. The following Specific Aims examine this central working hypothesis: Aim 1. Analyze the role of the electron transport chain in regulating cell surface F0F1 ATP synthase activity in gonadotropes. Aim 2. Determine the physiological importance of the ectoNTPDase CD39 in modulating GnRH action. Aim 3. Define the origin of F0F1 ATP synthase metabolites in the extracellular space.