Structure and Function of Dopamine Receptors

项目摘要 药物滥用通过中脑边缘多巴胺能通路诱导和促进多巴胺神经传递， 它改变了奖励相关的行为，并与药物成瘾的发展有关。安装 有证据表明，成瘾者在多巴胺水平和信号传导上表现出明显的缺陷， D2受体（D2 R），其有助于降低成瘾受试者对天然兴奋剂的敏感性， 使他们容易受到更强烈的药物刺激而产生欣快感。因此，D2 R提出了一个非常有前途的 临床前和临床研究都支持这一点。尽管D2 R激动剂 作为药物成瘾的治疗干预措施正在被深入研究，它们取得了巨大的成功 由于依从性和疗效差或与多巴胺能药物相关的副作用而受到阻碍。 D2 R的结构信息不仅可以揭示其信号转导机制，而且可以提供所需的信息 合理的药物设计。 作为G蛋白偶联受体（GPCR）家族的成员，D2 R以形成衍射性质而闻名 这些晶体对于通过X射线晶体学确定高分辨率结构至关重要。这笔赠款 该提案旨在为D2 R的结构和功能研究开发一个全面而强大的平台。到 为了实现这一目标，我们将致力于三个主要目标：1）开发优化的协议，获得纯化的 受体的量足以进行结晶尝试和功能研究，2）使用来自 目的1筛选最佳配体，用一系列高质量的配体评价受体-配体复合物的质量。 对照分析，和3）共结晶受体-配体复合物用于结构测定和建立受体-配体复合物。 包含用于功能研究的纳米盘，以及寻找高亲和力的纳米抗体或化合物， D2R该平台将涉及多个步骤，这些步骤紧密相连，并通过一个向前和 反向反馈系统 虽然在自然界中具有高度挑战性，但我们已经获得了一些可以表达的人D2 R构建体， 并在我们的实验室中纯化到每升生物质超过1毫克蛋白质，这对于 实现后续目标。本研究对多巴胺受体家族及 相关药物滥用研究：1）深入了解偏向激动，2）揭示变构调节的信息， 3)广泛筛选产生新的高亲和力D2 R配体，4）表征D2 R的机制 不同多巴胺受体亚家族之间的信号传导和配体选择，5）刺激基于结构的 药物设计，6）建立用于探索靶向D2 R的配体的多药理学的平台，以及7） 为GPCR/G蛋白复合物信号转导研究和理解GPCR/G蛋白复合物信号转导中的激活机制铺平了道路。 未来

Project Summary Drug abuse induces and facilitates dopamine neurotransmission through the mesolimbic dopaminergic pathway, which modifies reward-related behaviors and is associated with the development of drug addiction. Mounting evidence suggests that addicted individuals exhibited significant deficits on the level and signaling of dopamine D2 receptor (D2R), which contribute to decreased sensitivity of addicted subjects to natural reinforcers and predispose them to more potent drug stimulation of euphoria. Therefore, D2R presents a highly promising therapeutic target for drug abuse, as supported by both pre-clinical and clinical studies. Although D2R agonists are being intensively studied as therapeutic intervention for drug addiction, their success have been greatly hampered due to poor adherence and efficacy, or associated side effects related to dopaminergic medications. Structural information on D2R will not only reveal its signaling mechanisms, but also provide required information for rational drug design. As a member in the G protein-coupled receptor (GPCR) family, D2R is notorious to form diffraction-quality crystals that are essential for the determination of high-resolution structures by X-ray crystallography. This grant proposal aims to develop a comprehensive and robust platform for structural and functional studies of D2R. To achieve this goal, we will address three major aims: 1) develop optimized protocols for obtaining purified receptors in quantities sufficient for crystallization attempts and functional studies, 2) use stable receptors from Aim 1 to screen for optimal ligands and assess the quality of receptor-ligand complexes with an array of quality- control analyses, and 3) co-crystallize receptor-ligand complexes for structure determination and set up receptor- containing nanodiscs for functional studies as well as searching for high-affinity nanobodies or compounds for D2R. This platform will involve multiple steps that are closely interconnected and looped through a forward and backward feedbacks system. Although highly challenging in nature, we have acquired a few human D2R constructs that could be expressed and purified to more than 1 mg protein per liter of biomass in our laboratory, which is essential for the accomplishment of subsequent aims. The significance of this study is multi-fold on dopamine receptor family and related drug abuse studies: 1) gain insights into biased agonism, 2) reveal information on allosteric modulations, 3) screen extensively to produce new high-affinity ligands for D2R, 4) characterize the mechanisms of D2R signaling and ligand selection between different dopamine receptor subfamilies, 5) stimulate structure-based drug design, 6) establish the platform for exploring the polypharmacology of the ligands that target D2R, and 7) pave the road for GPCR/G protein complex signaling studies and understanding the activation mechanism in the future.