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途径。这些奖赏途径被纹状体中的神经递质多巴胺激活，多巴胺通过激活G蛋白发挥作用。G蛋白充当分子开关;它们被多巴胺受体"打开"，并被一类称为G蛋白信号调节器或RGS蛋白的蛋白质"关闭"。纹状体特异性RGS蛋白RGS9 - 2已被证明可以减少多巴胺通路激活剂可卡因的奖励效应和行为反应。该提案旨在确定RGS9 - 2抑制多巴胺信号传导的机制，以响应可卡因等成瘾药物。项目概述：腹侧纹状体中的多巴胺信号在多种药物成瘾中起着关键作用。一种新的纹状体特异性G蛋白信号转导调节因子（RGS）蛋白RGS9 - 2在体内调节多巴胺信号转导的能力使其成为开发成瘾治疗剂的有吸引力的靶点。虽然RGS9 - 2的RGS结构域可以使多巴胺受体下游的G蛋白失活的机制是明确的，但RGS9 - 2含有三个额外的结构域，每个结构域都涉及与其他细胞信号传导组分的相互作用。此外，纹状体中存在多种受RGS9 - 2差异调节的受体途径。该提案的第一个目标是定义RGS9 - 2调节多巴胺信号传导的不依赖于RGS的机制，并确定每个功能结构域在RGS9 - 2的整体细胞功能中的贡献。RGS9 - 2 G-γ样（GGL）结构域形成新型G蛋白异源三聚体的能力将使用核苷酸交换测定法进行生化测定。这些GGL介导的功能和其他RGS独立功能与RGS9 - 2活性的相关性将在细胞信号传导测定中使用缺乏确定结构域的截短和突变的RGS9 - 2构建体来确定。最后，还将在细胞信号传导测定中确定纹状体受体中RGS9 - 2调节的受体选择性。将使用稳态GTdR试验研究特异性机制，以确定G蛋白特异性和免疫沉淀的作用，并使用共聚焦显微镜研究受体-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.