Regulation of dopamine signaling by striatal RGS9-2; mechanisms and specificity

描述（由申请人提供）：相关性：滥用药物刺激大脑中与成瘾发展有关的奖励通路。这些奖赏通路是由纹状体中的神经递质多巴胺激活的，它通过激活g蛋白起作用。g蛋白作为分子开关；它们由多巴胺受体“开启”，由一类被称为g蛋白信号调节器（RGS）的蛋白质“关闭”。一种纹状体特异性RGS蛋白RGS9-2已被证明可以降低多巴胺通路激活剂可卡因的奖励效应和行为反应。本研究旨在确定RGS9-2在对可卡因等成瘾药物的反应中抑制多巴胺信号的机制。项目总结：腹侧纹状体多巴胺信号在多种药物成瘾中起关键作用。一种新的纹状体特异性g蛋白信号调节器（RGS）蛋白RGS9-2在体内调节多巴胺信号的能力使其成为成瘾疗法开发的一个有吸引力的靶点。虽然RGS9-2的RGS结构域可能使多巴胺受体下游的g蛋白失活的机制已经明确，但RGS9-2包含三个额外的结构域，每个结构域都与其他细胞信号传导成分的相互作用有关。此外，纹状体中存在多种受RGS9-2调控的受体通路。本研究的第一个目标是明确RGS9-2调节多巴胺信号的rgs独立机制，并确定RGS9-2各功能域在整体细胞功能中的贡献。RGS9-2 g - γ样（GGL）结构域形成新的g蛋白异源三聚体的能力将通过核苷酸交换法进行生化测定。这些ggl介导的功能和其他与RGS无关的功能与RGS9-2活性的相关性将在使用缺少定义结构域的截断和突变的RGS9-2构建体的细胞信号分析中确定。最后，RGS9-2调控纹状体受体的受体选择性也将在细胞信号分析中确定。特异性机制将通过稳态GTPase测定来确定g蛋白特异性的作用，免疫沉淀和共聚焦显微镜来确定受体- rgs蛋白相互作用的作用。

DESCRIPTION (provided by applicant): Relevance: Abused drugs stimulate reward pathways in the brain that are implicated in the development of addiction. These reward pathways are activated by the neurotransmitter dopamine in the striatum, which functions by activating G-proteins. G-proteins act as a molecular switch; they are turned "on" by dopamine receptors, and turned "off" by a class of proteins called Regulator of G-protein Signaling, or RGS proteins. A striatum-specific RGS protein, RGS9-2, has been shown to reduce the reward effects and behavioral responses of the dopamine pathway activator cocaine. This proposal seeks to define the mechanism of RGS9-2 inhibition of dopamine signaling in response to addictive drugs like cocaine. Project summary: Dopamine signaling in the ventral striatum plays a critical role in addiction to multiple agents. The ability of a novel striatal-specific Regulator of G-protein Signaling (RGS) protein, RGS9-2, to regulate dopamine signaling in vivo has made it an attractive target for the development of addiction therapeutics. Although the mechanism by which the RGS domain of RGS9-2 may deactivate G-proteins downstream of dopamine receptors is well defined, RGS9-2 contains three additional domains, each of which has been implicated in interactions with other cellular signaling components. Further, multiple receptor pathways exist in striatum that are differentially regulated by RGS9-2. The first goal of this proposal is to define RGS-independent mechanisms of RGS9-2 regulation of dopamine signaling and determine the contribution of each functional domain in the overall cellular function of RGS9-2. The ability of the RGS9-2 G-gamma-like (GGL) domain to form novel G-protein heterotrimers will be determined biochemically using nucleotide exchange assays. The relevance of these GGL-mediated functions and other RGS independent functions to RGS9-2 activity will be determined in cell signaling assays using truncated and mutated RGS9-2 constructs that lack defined domains. Finally, the receptor-selectivity of RGS9-2 regulation among striatal receptors will also be determined in cell signaling assays. The mechanism for specificity will be investigated using steady state GTPase assays to define the role of G-protein specificity and immunoprecipitation and confocal microscopy to define the role of receptor-RGS protein interactions.