Alterations in RGS9 Degradation Mechanisms Conferred by Opioids

阿片类药物引起的 RGS9 降解机制的改变

• 批准号: 7760044
• 负责人: Garret R. Anderson
• 金额: $ 1.4万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-15 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7760044
• 关键词: Acute; Affect; Amphetamines; Behavioral; Binding; Biological Assay; Brain; Cocaine; Complex; Data; Development; Distant; Dopamine; Dopamine Receptor; Drug Addiction; Drug effect disorder; Elements; Family; Functional disorder; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Protein Regulators; Hour; Knockout Mice; Knowledge; Lysine; Lysosomes; Maps; Mediating; Membrane; Messenger RNA; Molecular; Morphine; Motion; Movement; Movement Disorders; Mus; Nervous system structure; Neurites; Neurons; Nociception; Opioid; Opioid Receptor; Parkinson Disease; Pathway interactions; Pattern; Pharmaceutical Preparations; Physiologic pulse; Physiological Processes; Play; Process; Protein Binding; Proteins; Proteolysis; Pulse Rates; RGS Domain; Receptor Activation; Regulation; Rewards; Role; Route; Signal Pathway; Site-Directed Mutagenesis; Source; Subcutaneous Injections; Synaptic plasticity; Testing; Ubiquitin; Ubiquitination; Work; addiction; drug of abuse; inhibitor/antagonist; member; mutant; neuroprotection; neurotransmission; receptor; response; reward processing

DESCRIPTION (provided by applicant): Stimulation of G-protein coupled receptors (GPCR) is the mechanism of action by drugs of abuse such as cocaine and amphetamine (which exert their effects via dopamine receptors), and morphine (which acts on u-opioid receptors). Activation of these receptors is known to modulate such physiological processes as movement, nociception, and most critically in terms of abuse potential, the reward pathway. However, over stimulation of these receptors by drug action, leads to significant short-term and long-term changes within the dopamine and u-opioid GPCR signaling pathways that are as a result the critical determinants for the development of addiction. One such component of these pathways is the Regulator of G-protein Signaling 9 (RGS9), which has been shown to have a critical role in determining the behavioral response to the administration of these drugs of abuse. RGS9 serves as a negative regulator of dopamine and u-opioid action, and upon administration of drugs such as morphine, RGS9 protein levels increase rapidly and demonstrate lasting changes. This in turn can have a profound impact on the behavioral response elicited. It is the hypothesis of this proposal that alterations in RGS9 protein instability is a key determinant behind the volatility observed in RGS9 levels upon drug administration. The objective of this proposal therefore, is to investigate the molecular mechanisms regulating RGS9 degradation, and how these mechanisms are altered by drug action. Preliminary data suggests that ubiquitination/lysosomal targeting is a key component of RGS9 degradation, leading us to test this hypothesis by evaluating specific lysosome inhibitors and their ability to rescue RGS9 from degradation (Aim #1). Targeting to the lysosome via ubiquitin has been shown to be mediated by K63 poly-ubiquitination, and since RGS9 has been shown by us to be poly-ubiquitinated, we will test our hypothesis that this is through K63 linkage via the use of ubiquitin mutant constructs (Aim #1). Further, mapping of specific lysine residues of RGS9 that are ubiquitinated will be performed via site directed mutagenesis (Aim #2). Lastly, the role of alteration in these RGS9 degradation mechanisms detailed in the first two aims, will be evaluated with the administration of morphine in Aim #3, in an effort to delineate the source of RGS9 volatility that is observed with morphine action. The study as a whole therefore will serve to push our knowledge forward of the mechanisms regulating RGS9, a key component of the fundamental processes of reward and motion. It is only by understanding these mechanistic changes that advancement of treatment options will be possible for reward and motion dysfunctions such as drug addiction and movement disorders (i.e. Parkinson's).

描述（由申请人提供）：G蛋白偶联受体（GPCR）的刺激是滥用药物的作用机制，如可卡因和安非他明（通过多巴胺受体发挥作用）和吗啡（作用于μ-阿片受体）。已知这些受体的激活调节诸如运动、伤害感受的生理过程，并且最关键的是在滥用潜力方面，调节奖赏途径。然而，通过药物作用对这些受体的过度刺激导致多巴胺和μ-阿片样物质GPCR信号通路内的显著短期和长期变化，其结果是成瘾发展的关键决定因素。这些途径的一个这样的组分是G蛋白信号传导调节器9（RGS 9），其已被证明在确定对这些滥用药物的施用的行为反应中具有关键作用。RGS 9作为多巴胺和u-阿片类作用的负调节剂，并且在施用药物如吗啡后，RGS 9蛋白水平迅速增加并表现出持久的变化。这反过来又会对引发的行为反应产生深远的影响。该提议的假设是，RGS 9蛋白不稳定性的改变是给药后观察到的RGS 9水平波动性背后的关键决定因素。因此，本提案的目的是研究调节RGS 9降解的分子机制，以及这些机制如何通过药物作用改变。初步数据表明，泛素化/溶酶体靶向是RGS 9降解的关键组成部分，这使得我们通过评估特异性溶酶体抑制剂及其拯救RGS 9免于降解的能力来测试这一假设（目的#1）。通过泛素靶向溶酶体已被证明是由K63多聚泛素化介导的，并且由于RGS 9已被我们证明是多聚泛素化的，我们将通过使用泛素突变体构建体来测试我们的假设，即这是通过K63连接的（目标#1）。此外，将通过定点诱变进行泛素化的RGS 9的特异性赖氨酸残基的作图（目的#2）。最后，将在目标#3中用吗啡的施用来评价在前两个目标中详述的这些RGS 9降解机制中的改变的作用，以试图描绘在吗啡作用下观察到的RGS 9挥发性的来源。因此，这项研究作为一个整体将有助于推动我们对调节RGS 9机制的认识，RGS 9是奖励和运动基本过程的关键组成部分。只有通过理解这些机制变化，治疗方案的进步才有可能用于奖励和运动功能障碍，如药物成瘾和运动障碍（即帕金森氏症）。