D2 Receptor Induced Sensitization of Adenylate Cyclase

描述(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、药物滥用和帕金森氏症。D2多巴胺受体的急性激活抑制了循环AMP的积聚；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积聚。腺酰环化酶(AC)信号的这种异源敏化发生在体内和体外几个G？I/O偶联受体持续激活之后。本研究的总体目标是阐明D2样多巴胺受体持续激活后AC异源敏化的分子机制。以前的研究支持一种假说，即异源敏化需要激活G？I/O亚基，通过G？依赖机制诱导敏化。我们假设G？亚基通过直接和间接两种机制导致单个AC亚型的异源敏化。间接机制可能涉及蛋白质-蛋白质相互作用和G？S。这些研究的一般方法将是异源表达D2L多巴胺受体和特征良好的野生型或突变型AC(如AC1、AC2和AC5)，用于在独特的细胞背景下(即G蛋白亚单位缺失)进行完整的细胞实验。这一策略利用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。第一个具体目标将检验这样一种假设，即某些AC亚型的异源敏化涉及G？？-AC相互作用，并需要G？亚单位信令。这些研究将使用一系列AC突变体、独特的细胞模型、G？？小分子抑制剂。亚单位信号和纹状体神经元。第二个具体目标将确定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)来探索G？？以及G？S亚基在调节活细胞内基础和药物诱导的蛋白质-蛋白质相互作用中的作用。第三个具体目标是利用BIFC在神经细胞模型中鉴定和表征AC“敏化相互作用体”。这些研究将使用BIFC进行cDNA文库筛选，以在活细胞中鉴定致敏诱导的AC相互作用蛋白。拟议研究的完成将提供关于特定G蛋白亚基和新的蛋白质靶点的机制信息，最终可能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.