Identification of the AC5 sensitization interactome using BiFC

使用 BiFC 鉴定 AC5 致敏相互作用组

• 批准号: 8510919
• 负责人: Carmen W. Dessauer
• 金额: $ 19.79万
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8510919
• 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，包括cAMP过调和AC的异源致敏。这种适应性反应与几种精神和神经系统疾病有关。先前的研究支持一种假设，即G？i/o偶联受体促进G？和G ? ?亚单位在百日咳毒素敏感的方式，诱导敏化通过一个尚未可知的机制。我们假设药物诱导的蛋白质与AC的相互作用是AC反应增强的原因。以前的研究已经检查了密切相关的蛋白质或建立的AC相互作用伙伴，阻止了真正新机制的发现。因此，一种前所未有的方法将被用于识别神经元细胞模型中腺苷酸环化酶5型（AC5）的“致敏相互作用组”。本研究将利用双分子荧光互补技术（BiFC）进行cDNA文库筛选，以鉴定活细胞中致敏诱导的AC5相互作用蛋白。特异性am1将构建并表征AC5药物性bbc的神经元细胞模型。这些研究将使用稳定表达工程AC5融合结构的CAD细胞，该结构能够与适当的结合伙伴进行荧光互补。特异性目标2构建基于逆转录病毒的潜在AC5结合伙伴的bbifc cDNA文库，用于FACS筛选。Specific aim 3应对药物性BiFC进行初级和二级筛查。这些研究将用逆转录病毒cDNA文库感染神经元细胞模型，然后用G？i/o受体激动剂诱导AC活性的异源增敏。揭示药物诱导的BiFC的细胞将使用FACS进行鉴定，并分离进行cDNA扩增。Specific aim 4将启动一系列生化和功能研究，以表征代表AC5“致敏相互作用组”的相互作用蛋白。这些目标的预期结果是鉴定和表征新的AC5相互作用蛋白相关