Molecular Mechanisms of Ontogenesis of K-Opioid Receptors

本部分将继续阐明kappa“个体发生”的分子机制。 阿片受体(KOR)，即发育阶段和分化过程中KOR蛋白的产生 神经元。在以前的资金周期中的研究已经为控制描绘了几条调控途径 在发育阶段KOR mRNA的产生，主要是转录调控。这就结束了 第一阶段研究重点是KOR的霍尔标志物KOR mRNA合成的调控 神经发生涉及维甲酸(维生素A)和一氧化氮的信号通路，并需要 KOR基因调控区的染色质重塑，以及在表观遗传控制方面的更新发现。 重要的是，KOR的个体发育似乎受转录后转录控制，最关键的是 信使核糖核酸的稳定性、转运和翻译等机制。此续订组件将重点放在 翻译机制从我们的初步研究延伸到Netrin-1是一种 KOR的翻译刺激因子，以及作为KOR的翻译抑制因子的Grb7。 两个具体目标是： 1.阐明Netrin-1/Grb7途径激活KOR翻译的机制。我们将重点关注i) Grb7对KOR翻译起始的调控，包括其分子和生化研究 特征和功能结构域，以及可被激活的Grb7结合的KOR RNA序列 Netrin-1，以及ii)Grb7靶向的特异翻译起始步骤，包括起始因子和 KOR mRNA的亚细胞分布和可能的循环。 2.Grb7/Netrin-1信号与KOR个体发生的药理学和生理学相关性我们会的) 进行功能得失研究以验证Netrin-1/Grb7在KOR个体发生中的相关性 使用干细胞和原代神经元，以及ii)检查KOR合成在 神经元的活动，如在特定的疼痛循环中，在神经损伤期间，或作为应激反应。

This component will continue elucidating the molecular mechanisms underlying "ontogenesis" of kappa opioid receptor (KOR), i.e. production of KOR protein during developmental stages and differentiating neurons. Studies in the previous funding cycles have delineated several regulatory pathways for the control of KOR mRNA production during developmental stages, principally transcriptional control. This concludes the first phase of studies focusing on the regulation of KOR mRNA synthesis, a hall markd of KOR neurogenesis involving signaling pathways of retinoic acid (vitamin A) and nitric oxide and requires chromatin remodeling of KOR gene regulatory regions, as well as more recent findings in epigenetic control. Importantly, ontogenesis of KOR appears to be controlled, most crucially, by post-transcriptional mechanisms such as mRNA stability, transport and translation. This renewal component will focus on translational mechanism by extending from our preliminary studies that have identified Netrin-1 as a translational stimulator for KOR, and Grb7 as a translational represser of KOR. Two specfiic aims are: 1. To elucidate the mechanism that activates KOR translation via Netrin-1/Grb7 pathway. We will focus on i) regulation of translational initiation of KOR by Grb7, including studies of its molecular and biochemical features and funcitonal domains, and KOR RNA sequences bound by Grb7 which can be activated by Netrin-1, and ii) specific translational initiation step that is targeted by Grb7 including initiation factors and subcellular distribution and possible circularization of KOR mRNA. 2. Pharmacological and physiological relevance of Grb7/Netrin-1 signaling to KOR ontogenesis. We will i) perform gain- and loss-of-function studies to validate the relevance of Netrin-1/Grb7 in KOR ontogenesis using stem cells and primary neurons, and ii) examine the physiological relevance of KOR synthesis in neuronal activity such as in a specific pain circiitry and during nerve injury or as a stress response.