Non-canonical signaling of retinoic acid by Cellular Retinoic Acid Binding Protein 1

摘要：全反式维甲酸(AtRA)是维生素A通过维甲酸作用的活性代谢物 受体(RARs)调节基因表达。越来越多的研究报告RAR独立(非规范) AtRA信号在各种生物系统中的活性。重要的是，遗传学研究已经证实，细胞 维甲酸结合蛋白1(Crabp1)介导这种atRA的非规范活性，如快速调节 干细胞周期调控中的细胞外信号调节激酶(ERK)激活。最近，我们已经确定了 Crabp1的另一个信号靶点是钙(Ca~(2+))-钙调蛋白依赖性蛋白激酶II(CaMKII)。CaMKII是 一种与多种细胞功能有关的重要的激酶。该提案中的初步数据支持 Crabp1-CaMKII信号体在调节维生素A营养作用中的生理学意义 调节细胞信号传递能力的内分泌。我们的初步数据显示CaMKII发生了戏剧性的变化 Crabp1基因敲除(CKO)小鼠运动神经元的活性。已识别的人类基因数据显著减少 Crabp1在某些运动神经元疾病中的表达，进一步支持了生理和病理 与Crabp1的相关性。主要假设是Crabp1信号体编排了一部小说(RAR- 独立)严格调节重要细胞内信号激活的全反式维甲酸反应机制 组件，如CaMKII和ERK。我推测这个Crabp1-CaMKII信号(这是重点 建议)在调节运动神经元活动方面发挥作用。我的提议的目的是阐明这种机制 这个Crabp1-CaMKII的信号。为了实现这一目标，提出了两个目标。目标1将描述 用分子、生化和结构生物学方法研究Crabp1和CaMKII的确切分子关系 接近了。全反式维甲酸和我们文库中的化合物(CRABP1选择性和RAR非依赖性)对血管内皮细胞生长的影响 这种分子关系也将被确定。AIM 2将功能验证Crabp1在调节CaMKII中的作用 在钙调节的神经元功能的生理相关细胞背景下的活动。这将是 通过使用)重组的神经元细胞系来分子剖析所需的关键元件来完成 对于CaMKII信号和b)病毒拯救原代CKO运动神经元中Crabp1的表达，随后 Crabp1-CaMKII途径的功能验证。功能分析包括：1)CaMKII的磷酸化 底物AMPAR、CREB和突触素。2)细胞内钙离子浓度的测量 神经元活动/放电(CaMKII活性的已知输出)。这项提议的意义在于提供了第一个 维生素A/atRA调节CaMKII活性和ATRA的新的非典型信号的分子理解 起到预防某些人类疾病的作用。在营养/内分泌方面，这项提议 阐述了atRA(一种内分泌和必需的营养物质)和一种重要的钙信号之间的新的相互作用 分子，CaMKII。这一结果将为人们深入了解维生素A的复杂性提供依据 信号转导与人类疾病新型维甲酸治疗药物的开发。

Abstract: All-trans retinoic acid (atRA) is an active metabolite of vitamin A that acts through retinoic acid receptors (RARs) to regulate gene expression. Increasing studies report RAR-independent (non-canonical) activity of atRA signaling in various biological systems. Importantly, genetic studies have established that cellular retinoic acid binding protein 1 (Crabp1) mediates this atRA non-canonical activity, such as rapid modulation of extracellular signal–regulated kinase (ERK) activation in stem cell cycle control. Recently, we have identified another signaling target of Crabp1, calcium (Ca2+)-calmodulin dependent protein kinase II (CaMKII). CaMKII is an important kinase implicated in numerous cellular functions. Preliminary data in this proposal support the physiological relevance of the Crabp1-CaMKII signalsome in mediating the action of vitamin A as a nutrient and an endocrine that modulates cellular signaling capacity. Our preliminary data reveal dramatically altered CaMKII activity in Crabp1 knockout (CKO) mouse motor neurons. Human genetic data identified significantly reduced Crabp1 expression in certain motor neuron diseases, further supporting the physiological and pathological relevance for Crabp1. The principal hypothesis is that the Crabp1-signalsome orchestrates a novel (RAR- independent) atRA-responsive mechanism that tightly regulates activation of important intracellular signaling components such as CaMKII and ERK. I hypothesize that this Crabp1-CaMKII signalsome (the focus of this proposal) plays a role in modulating motor neuron activity. The goal of my proposal is to elucidate the mechanism of this Crabp1-CamKII signalsome. To achieve this goal, two aims are proposed. Aim 1 will characterize the exact molecular relationship between Crabp1 and CaMKII using molecular, biochemical, and structural biology approaches. The effect of atRA and compounds from our library (Crabp1-selective and RAR-independent) on this molecular relationship will also be determined. Aim 2 will functionally validate Crabp1 in regulating CaMKII activity in a physiologically relevant cellular context of Ca2+-regulated neuronal functions. This will be accomplished by employing a) reconstituted neuron cell lines to molecularly dissect critical elements required for CaMKII signaling and b) viral rescue of Crabp1 expression in primary CKO motor neurons, followed by functional validation of this Crabp1-CaMKII pathway. Functional assays include 1) phosphorylation of CaMKII substrates AMPAR, CREB, and synapsin. 2) Measurement of intracellular Ca2+ concentrations that mediate neuron activity/firing (a known output of CaMKII activity). The significance of this proposal is to provide the first molecular understanding of a new non-canonical signaling of vitamin A/atRA that modulates CaMKII activity and plays a role in protecting against certain human diseases. In a nutrition/endocrine context, this proposal addresses a new cross-talk between atRA (an endocrine and essential nutrient) and an important Ca2+ signaling molecule, CaMKII. The results will provide insight on the increasingly recognized complexity in vitamin A signaling and the development of novel retinoid therapeutics for human disease.