D2 Receptor Induced Sensitization of Adenylate Cyclase

描述（由申请人提供）：D2多巴胺受体与神经精神和神经疾病有关，包括精神分裂症、药物滥用和帕金森病。急性激活D2多巴胺受体抑制AMP循环积累；然而，D2多巴胺受体的持续激活增强了随后药物刺激的循环AMP积累。腺苷酸环化酶（AC）信号的异源致敏发生在几个G？体外和体内I /o偶联受体。本研究计划的总体目标是阐明d2样多巴胺受体持续激活后AC异源致敏的分子机制。先前的研究支持一种假设，即异源致敏需要激活G？i/o亚基通过G??诱导致敏端依赖机制。我们假设G?？亚基通过直接和间接的机制导致单个AC亚型的异源致敏。间接机制可能包括蛋白质-蛋白质相互作用和G?s。这些研究的一般方法是在独特的细胞背景（即G蛋白亚基缺乏）下进行完整细胞实验，将异源D2L多巴胺受体与具有良好特征的野生型或突变型ACs（如AC1、AC2和AC5）一起表达。该策略利用了最近发现的分子和细胞工具来研究G蛋白信号以及新的荧光技术。第一个具体目的是验证AC选择异种致敏性涉及G?？-AC相互作用，需要G?？亚基的信号。这些研究将使用一系列AC突变体、独特的细胞模型、G?？亚单位信号和纹状体神经元。第二个特定目标将确定G蛋白亚基在调节受体- ac和AC-AC相互作用中的作用和要求。这些实验将利用双分子荧光互补（BiFC）来探测G?？和G ?S亚基在活细胞中调节基础和药物诱导的蛋白相互作用。第三个具体目标是在神经元细胞模型中使用bic识别和表征AC“致敏相互作用组”。这些研究将使用BiFC进行cDNA文库筛选，以鉴定活细胞中敏化诱导的AC相互作用蛋白。该研究的完成将提供关于特定G蛋白亚基和新蛋白靶点的机制信息，最终可用于防止体内异源致敏的发展和表达。

DESCRIPTION (provided by applicant): D2 dopamine receptors have been implicated in neuropsychiatric and neurologic disorders including schizophrenia, drug abuse, and Parkinson's disease. Acute activation of D2 dopamine receptors inhibits cyclic AMP accumulation; however, persistent activation of D2 dopamine receptors enhances subsequent drug-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylyl cyclase (AC) signaling occurs following persistent activation of several G?i/o-coupled receptors in vitro and in vivo. The overall objective of this research proposal is to elucidate the molecular mechanisms involved in heterologous sensitization of AC following persistent activation of D2-like dopamine receptors. Previous studies support a hypothesis that heterologous sensitization requires the activation of G?i/o subunits to induce sensitization through a G??-dependent mechanism. We hypothesize that G?? subunits lead to heterologous sensitization of individual AC isoforms through both direct and indirect mechanisms. The indirect mechanisms may involve protein-protein interactions as well as G?s. The general approach for these studies will be to express heterologously D2L dopamine receptors together with well characterized wild-type or mutant ACs (e.g. AC1, AC2, and AC5) for intact cell experiments in unique cellular backgrounds (i.e., G protein subunit deficient). This strategy takes advantage of recently discovered molecular and cellular tools to study G protein signaling as well as novel fluorescent technologies. The first specific aim will test the hypothesis that heterologous sensitization of select isoforms of AC involves G??-AC interactions and requires G?? subunit signaling. These studies will use a series of AC mutants, unique cellular models, small molecule inhibitors of G?? subunit signaling, and striatal neurons. The second specific aim will determine the roles and requirements for G protein subunits in modulating receptor-AC and AC-AC interactions. These experiments will use bimolecular fluorescence complementation (BiFC) to probe the specific role of G?? and G?s subunits in modulating basal and drug-induced protein-protein interactions in living cells. The third specific aim will identify and characterize the AC "sensitization interactome" using BiFC in a neuronal cell model. These studies will use BiFC to perform cDNA library screening to identify sensitization-induced interacting proteins of AC in living cells. Completion of the proposed studies will deliver mechanistic information regarding specific G protein subunits and new protein targets that could ultimately be used to prevent the development and expression of heterologous sensitization in vivo. PUBLIC HEALTH RELEVANCE: Understanding the molecular mechanisms responsible for D2 dopamine receptor-induced sensitization of adenylyl cyclase has implications in a variety of diseases including schizophrenia, Parkinson's disease, and drug abuse. Sensitization of adenylyl cyclase signaling occurs following persistent activation of several G?i/o-coupled receptors. Thus, the information discovered here is also relevant to many G protein-coupled receptors that are targets of drugs used to treat pain, depression, and Alzheimer's disease (e.g., opioid, serotonin, and muscarinic receptors).