Identification of the AC5 sensitization interactome using BiFC

描述(由申请人提供)：G？I/O偶联受体的持续激活导致腺酰环化酶(AC)信号的增强，该信号已被用许多不同的名称描述，包括cAMP超调和AC的异源敏化。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G？i/o偶联受体持续激活促进G？i/o偶联受体解离/重排的假说。和G？以百日咳毒素敏感的方式，通过一种未知的机制诱导致敏。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已建立的AC相互作用伙伴，阻止了真正新机制的发现。因此，一种史无前例的方法将被用来在神经细胞模型中鉴定腺酰环化酶5(AC5)的“敏化相互作用组”。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AC5的致敏诱导相互作用蛋白。特异性AM-1将构建和表征药物诱导的AC5的BIFC的神经细胞模型。这些研究将使用稳定表达工程AC5融合结构的CAD细胞，该融合结构能够与适当的结合伙伴进行荧光互补。特异性目的2构建基于逆转录病毒的潜在AC5结合伙伴的BIFC文库，用于FACS的筛选。具体目标3应对药物引起的BIFC进行一次和二次筛查。这些研究将用逆转录病毒cDNA文库感染神经细胞模型，然后用G？I/O受体激动剂诱导AC活性的异源敏化。发现药物诱导的BIFC的细胞将通过FACS进行鉴定，并分离出来进行cDNA扩增。“特定目标4”将启动一系列生化和功能研究，以表征代表AC5“敏化相互作用体”的相互作用蛋白。这些目标的预期结果是鉴定和鉴定与ac5相互作用的新蛋白。 对异源致敏。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方法，并有可能为体内研究提供药物靶点。

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.