Alterations in RGS9 Degradation Mechanisms Conferred by Opioids

描述(由申请人提供)：G蛋白偶联受体的刺激是滥用药物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u-阿片受体)的作用机制。众所周知，这些受体的激活可以调节运动、伤害性感受等生理过程，最关键的是，就滥用潜力而言，是奖赏途径。然而，药物作用对这些受体的过度刺激会导致多巴胺和u-阿片GPCR信号通路的短期和长期显著变化，从而导致成瘾发展的关键决定因素。这些途径中的一个这样的组成部分是G蛋白信号转导9(RGS9)的调节因子，它已被证明在决定对这些药物滥用的行为反应中具有关键作用。RGS9是多巴胺和u-阿片类药物作用的负性调节因子，在使用吗啡等药物后，RGS9蛋白水平迅速升高，并表现出持久的变化。这反过来会对引起的行为反应产生深远的影响。这一假设认为，RGS9蛋白不稳定性的改变是给药后RGS9水平波动的关键决定因素。因此，这项建议的目的是研究调节RGS9降解的分子机制，以及这些机制是如何被药物作用改变的。初步数据表明，泛素化/溶酶体靶向是RGS9降解的关键组成部分，这导致我们通过评估特定的溶酶体抑制剂及其将RGS9从降解中拯救出来的能力来检验这一假说(目标#1)。通过泛素靶向溶酶体已被证明是由K63多泛素化介导的，由于我们已经证明RGS9是多泛素化的，我们将检验我们的假设，即这是通过使用泛素突变结构来连接K63(目标#1)。此外，泛素化的RGS9的特定赖氨酸残基的图谱将通过定点突变(目标2)进行。最后，前两个目标中详述的改变在这些RGS9降解机制中的作用，将通过在目标3中给药吗啡来评估，以努力描绘通过吗啡作用观察到的RGS9挥发性的来源。因此，作为一个整体，这项研究将有助于推动我们对RGS9调控机制的了解，RGS9是奖励和运动基本过程的关键组成部分。只有通过了解这些机制的变化，才有可能改进奖赏和运动功能障碍的治疗方案，如药物成瘾和运动障碍(即帕金森氏症)。

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).