Identification of the AC5 sensitization interactome using BiFC

使用 BiFC 鉴定 AC5 致敏相互作用组

• 批准号: 8703794
• 负责人: Carmen W. Dessauer
• 金额: $ 23.83万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-19 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8703794
• 关键词: Acute; Address; Adenosine; Adenylate Cyclase; Agonist; Binding; Biochemical; Biological Assay; Brain; Cell model; Cells; Cellular Assay; Chronic; Complement; Complementary DNA; Cyclic AMP; DNA Sequence Rearrangement; Dissociation; Dopamine; Dopamine D2 Receptor; Drug Targeting; Drug abuse; Engineering; Fluorescence; Future; G-Protein-Coupled Receptors; Goals; Human; Investigation; Life; Link; Messenger RNA; Molecular; Muscarinics; Names; Neurologic; Neurons; Opioid Receptor; Outcome; Pain management; Parkinson Disease; Pathway interactions; Pertussis Toxin; Pharmaceutical Preparations; Physical Dependence; Preclinical Drug Evaluation; Proteins; Receptor Activation; Receptor Signaling; Research; Retroviridae; Schizophrenia; Series; Serotonin; Signal Transduction; Validation; Venus; Work; adenylyl cyclase type V; base; cDNA Library; dopamine D2L receptor; drug of abuse; in vivo; insight; mutant; novel; prevent; public health relevance; receptor; receptor coupling; research study; response; screening; stable cell line; vector

DESCRIPTION (provided by applicant): Persistent activation of G?i/o-coupled receptors leads to enhanced adenylyl cyclase (AC) signaling that has been described using many different names, including cAMP overshoot and heterologous sensitization of AC. This adaptive response has been implicated in several psychiatric and neurological conditions. Previous studies support a hypothesis that persistent activation of G?i/o-coupled receptors promotes the dissociation/rearrangement of G? and G?? subunits in a pertussis toxin-sensitive manner that induces sensitization through a yet unknown mechanism. We hypothesize that drug-induced protein interactions with AC are responsible for the enhanced AC response. Previous studies have examined closely-related proteins or established AC interacting partners preventing the discovery of truly novel mechanisms. Thus, an unprecedented approach will be used to identify the "sensitization interactome" of adenylyl cyclase type 5 (AC5) in a neuronal cell model. These studies will use Bimolecular Fluorescence Complementation (BiFC) to perform cDNA library screening to identify sensitization-induced interacting proteins of AC5 in living cells. Specific am 1 will construct and characterize a neuronal cellular model for drug-induced BiFC of AC5. These studies will use CAD cells stably expressing an engineered AC5 fusion construct capable of fluorescence complementation with appropriate binding partners. Specific aim 2 shall construct the retrovirus-based BiFC cDNA library of potential AC5 binding partners for FACS screening. Specific aim 3 shall execute both primary and secondary screening for drug-induced BiFC. These studies will infect the neuronal cell model with the retroviral cDNA library followed by treatment with a G?i/o receptor agonist to induce heterologous sensitization of AC activity. Cells revealing drug-induced BiFC will be identified using FACS and isolated for cDNA amplification. Specific aim 4 will initiate a series of biochemical and functional studies to characterize those interacting proteins that represent the AC5 "sensitization interactome." The anticipated outcome of these aims is the identification and characterization of novel AC5 interacting proteins relevant to heterologous sensitization. The impact of these scientific outcomes is substantial and will address a long-standing scientific question, develop a novel methodological approach, and have the potential to provide drug targets for in vivo studies.

描述（由申请人提供）：G？I/O偶联受体导致腺苷酸环化酶（AC）信号传导增强，其已经使用许多不同的名称描述，包括cAMP过冲和AC的异源致敏。这种适应性反应与几种精神和神经疾病有关。以往的研究支持一个假设，持续激活G？I/O偶联受体促进G？而G？在百日咳毒素敏感的方式，通过一个未知的机制诱导致敏的亚基。我们推测药物诱导的蛋白质与AC的相互作用是增强AC反应的原因。以前的研究已经检查了密切相关的蛋白质或建立AC相互作用的合作伙伴，防止发现真正的新机制。因此，一个前所未有的方法将被用来确定在神经元细胞模型中的腺苷酸环化酶5型（AC 5）的“致敏相互作用组”。这些研究将使用双分子荧光互补（BiFC）进行cDNA文库筛选，以鉴定活细胞中AC 5的致敏诱导相互作用蛋白。特异性am 1将构建和表征用于药物诱导的AC 5的BiFC的神经元细胞模型。这些研究将使用稳定表达工程化AC 5融合构建体的CAD细胞，所述工程化AC 5融合构建体能够与适当的结合配偶体进行荧光互补。具体目标2应构建基于逆转录病毒的潜在AC 5结合配偶体的BiFC cDNA文库，用于FACS筛选。具体目标3应针对药物诱导的BiFC进行一级和二级筛查。这些研究将感染的神经元细胞模型与逆转录病毒的cDNA文库，然后用G？I/O受体激动剂诱导AC活性的异源致敏。将使用FACS鉴定显示药物诱导的BiFC的细胞，并分离用于cDNA扩增。具体目标4将启动一系列生物化学和功能研究，以表征代表AC 5致敏相互作用组的那些相互作用蛋白。“这些目标的预期结果是鉴定和表征新的AC 5相互作用蛋白， 异源致敏这些科学成果的影响是巨大的，将解决一个长期存在的科学问题，开发一种新的方法学途径，并有可能为体内研究提供药物靶点。