Proteomic Characterization of the Sigma-1 Receptor and Its Signaling Complex

描述(由申请人提供)：对sigma-1受体如何在蛋白质水平上调节，以及这如何影响其功能的基本理解仍然缺乏。这一点很重要，因为sigma受体代表了一类新的蛋白质，当被可卡因和甲基苯丙胺等精神刺激剂激活时，有助于大脑的短期和长期适应。我们的长期目标是阐明控制Sigma受体激活的调控机制，作为充分了解Sigma受体功能的前提。这项R03应用的总体目标，是朝着实现我们的长期目标迈出的第一步，目的是充分表征Sigma-1受体及其“信号复合体”。这一应用的中心假设是，sigma-1受体利用相互作用的蛋白质网络将配体结合事件转化为生物作用，该信号网络受到翻译后蛋白质修饰和差异蛋白质-蛋白质相互作用的调节。这项研究的理论基础是，一旦我们了解了Sigma-1受体信号是如何控制的，就有可能更彻底地研究Sigma-1受体对加性行为的调节。由于Sigma-1受体与各种生物学过程相关，这一知识可能会导致成功的策略，特别是靶向结构(和功能)不同的异构体。这项拟议的研究与美国国立卫生研究院的任务相关，因为它将扩大我们的基本知识，并潜在地改善人类健康。为了检验我们的中心假设并实现这一应用的目标，将追求两个特定的目标：1)在存在和不存在配体的情况下识别sigma-1受体翻译后修饰；2)在存在和不存在配体的情况下识别sigma-1受体相互作用的蛋白质。我们将通过使用量身定制的纯化方案和特定的标记策略，以及优化的高效液相色谱梯度和最先进的质谱仪来实现这一点。这项拟议的研究具有重要意义，因为它将使我们更好地从根本上理解一类新的膜结合受体，并扩大我们对该受体如何受到滥用药物调控的理解。公共卫生相关性：Sigma-1受体，潜在的药物成瘾和滥用的重要治疗靶点，将在拟议的研究中全面描述。这项拟议的研究与公共健康相关，因为研究结果有望让人们更好地理解如何操纵这些蛋白质来治疗药物滥用。

DESCRIPTION (provided by applicant): A fundamental understanding of how the sigma-1 receptor is regulated at the protein level, and how this impacts its function, is still missing. This is important because the sigma receptors represent a novel class of proteins that when activated by psychostimulants, such as cocaine and methamphetamine, contribute to both short- and long-term adaptations in the brain. Our long-term goal is to elucidate the regulatory mechanisms controlling activation of sigma receptors as a prerequisite to fully understanding sigma receptor function. The overall objective of this R03 application, which is a first step toward attainment of our long-term goal, is to fully characterize the sigma-1 receptor and its "signaling complex." The central hypothesis of this application is that the sigma-1 receptor utilizes a network of interacting proteins to translate ligand binding events into biological action and this signaling network is subject to regulation by post-translational protein modifications and differential protein-protein interactions. The rationale for the proposed research is that it will be possible to study sigma-1 receptor regulation of additive behaviors more thoroughly once we understand how its signaling is controlled. Since the sigma-1 receptor is associated with various biological processes, this knowledge will potentially lead to successful strategies for targeting structurally (and functionally) diverse isoforms specifically. The proposed research is relevant to NIH's mission because it will expand our basic knowledge and potentially improve the human health. Two specific aims will be pursued in order to test our central hypothesis and accomplish the objective of this application: 1) Identify sigma-1 receptor post-translational modifications in the presence and absence of ligands; and 2) Identify sigma-1 receptor interacting-proteins in the presence and absence of ligands. We will accomplish this by using tailored purification schemes and specific labeling strategies in conjunction with optimized HPLC gradients and state-of-the art mass spectrometry instrumentation. The proposed research is significant because it will lead to a better fundamental comprehension of a novel class of membrane bound receptors and expand our understanding of how the receptor is regulated by drugs of abuse. PUBLIC HEALTH RELEVANCE: Sigma-1 receptors, potentially important therapeutic targets for drug addiction and abuse, will be comprehensively characterized in the proposed studies. The proposed research is relevant to public health because the results are expected to lead to a better understanding of how these proteins can be manipulated to treat substance abuse.