The role of RGS12 in differential modulation of G protein versus beta-arrestin signaling downstream of the kappa opioid receptor

Kappa阿片受体(KOR)有助于调节对压力的反应，但也与发展和/或 维护慢性疼痛、药物成瘾、焦虑和抑郁等健康障碍。KOR激动剂已经显示出 在改善这些疾病方面有希望，但受到烦躁副作用的限制。朝鲜民主主义人民共和国的好处 激动剂(如止痛)被认为主要由G蛋白信号转导，而β-arrestin 信号传递被认为是其有害副作用(如烦躁不安)的核心。然而，机制(S) KOR下游的这些信号是通过什么来调节的，目前仍在阐明。的监管者 G蛋白信号转导是G蛋白偶联受体后加速信号终止的细胞内蛋白 激活。RGS12是这个蛋白质家族的复杂成员，至少有五个不同的结构域相互作用 具有G蛋白依赖和非依赖信号通路的组成部分。我们最近报道说 RGS12在腹侧纹状体(VSTR)中丰富，而整体RGS12消融会降低运动反应 多巴胺(DA)--调节精神刺激剂。我们的数据与增强的DA转运体(DAT)相关 RGS12基因缺失小鼠的VSTR，而不是背侧纹状体(DSTR)的表达和功能。RGS12丢失 (尤其是作为GAI/O导向的GTP酶加速蛋白)可能间接增加DAT的表达/功能 通过消除KOR下游的负调控，鉴于KOR激动剂已知会增加DAT 表面表达和摄取功能。为了支持这一概念，扩展的DAT功能和简化的 KOR拮抗后，由RGS12缺失引起的AMPH刺激的运动均被逆转。我们也 在RGS12缺失和β-arrestin2缺失小鼠品系的vstr和dstr中发现KOR表达升高。 RGS12过表达增加β-arrestin对激活的KOR的募集--这一效应在 百日咳毒素介导的Gi/o抑制或突变对RGS12的GA相互作用结构域，提示 G蛋白非依赖性机制。这些发现与我们的最新数据一致，即RGS12基因缺失的小鼠 表现出减弱的KOR激动剂诱导的条件性位置厌恶，被认为是β-arrestin依赖的行为。 总而言之，我们的数据强调了RGS12作为一种新的、差异化的G蛋白依赖的调节因子的作用 KOR激活下游的与AND无关的信号，这一调节可能被利用 从药理上讲，有助于将KOR介导的信号从有害结果转变为有益结果。 我们的第一个目标是确定RGS12调节G蛋白的特定神经元群体。 依赖和独立的KOR信号，测试RGS12操作以调制KOR和的假设 DAT在表达KOR和DAT的中枢神经系统神经元中起特异性作用，也在脊髓水平起作用 电源线。我们的第二个目标是描述产生选择性功能相互作用的分子决定因素。 在RGS12和韩国之间。成功地实现这两个目标将提供关键的临床前数据供考虑 RGS12是未来参与KOR信号转导的止痛和抗成瘾治疗的有效靶点。

The kappa opioid receptor (KOR) helps mediate responses to stress, yet is also implicated in developing and/or maintaining health disorders of chronic pain, drug addiction, anxiety and depression. KOR agonists have shown promise in ameliorating these disorders, but are limited by dysphoric side effects. Beneficial effects of KOR agonists (e.g., analgesia) are considered predominantly mediated by G protein signaling, whereas β-arrestin signaling is considered central to their detrimental side effects (e.g., dysphoria). However, the mechanism(s) by which these signals downstream of KOR are regulated are still being elucidated. The Regulators of G protein Signaling are intracellular proteins that accelerate signal termination after G protein-coupled receptor activation. RGS12 is a complex member of this protein family, with at least five different domains that interact with components of both G protein-dependent and -independent signaling pathways. We recently reported that RGS12 is enriched in the ventral striatum (vSTR), and global Rgs12 ablation decreases locomotor responses to dopamine (DA)-modulating psychostimulants. Our data correlate with the augmented DA transporter (DAT) expression and function in the vSTR, but not dorsal striatum (dSTR), seen in RGS12-null mice. Loss of RGS12 (especially as a Gai/o-directed GTPase-accelerating protein) may indirectly increase DAT expression / function by removing negative regulation downstream of KOR, given that KOR agonists are known to increase DAT surface expression and uptake function. Supporting this notion, the augmented DAT function and reduced AMPH-stimulated locomotion caused by RGS12 loss are both reversed following KOR antagonism. We also found elevated KOR expression in the vSTR, but not dSTR, of RGS12-null and β-arrestin2-null mouse strains. RGS12 over-expression augments β-arrestin recruitment to activated KOR – an effect preserved following pertussis toxin-mediated Gi/o inhibition or mutation to the Ga-interacting domains of RGS12, suggesting a G protein-independent mechanism. These findings are consistent with our newest data that RGS12-null mice exhibit attenuated KOR agonist-induced conditioned place aversion, considered β-arrestin-dependent behavior. Collectively, our data highlight a role for RGS12 as a novel, differential regulator of both G protein-dependent and -independent signaling downstream of KOR activation, a regulation that may be exploitable pharmacologically to help shift KOR-mediated signaling to beneficial outcomes and away from detrimental ones. Our first aim is to determine the specific neuronal populations within which RGS12 acts to modulate G protein- dependent and -independent KOR signaling, testing hypotheses that RGS12 operates to modulate KOR and DAT function specifically in KOR- and DAT- expressing CNS neurons, and also operates at the level of the spinal cord. Our second aim is to delineate the molecular determinants that engender selective functional interactions between RGS12 and KOR. Success in pursuit of these two aims will provide key pre-clinical data for considering RGS12 a valid target for future analgesic and anti-addiction therapeutics that engage KOR signal transduction.