CB1 Receptor-Mediated Tyrosine Phosphorylation of FAK and ERK in Neurons

描述（由申请人提供）：这个Kirschstein-NRSA博士后奖学金（F32）将为乔治道尔顿提供研究培训博士的指导下。Allyn Howlett在生理学和药理学系在维克森林大学健康科学，这将使他发展成为一个成功的，独立的科学家。本申请中拟议研究的长期目标是表征CB 1受体（CB 1 R）介导的调节神经元中蛋白质酪氨酸（Tyr）磷酸化的信号传导事件。道尔顿博士已经确定，大麻素激动剂激活CB 1受体，以诱导N18 TG 2（N18）神经母细胞瘤细胞中粘着斑激酶（FAK）和细胞外信号调节激酶（ERK）的Tyr磷酸化。这些发现形成了本提案中两个具体目标的调查基础，这将涉及CB 1 Rs调节N18细胞中FAK和ERK的Tyr磷酸化的机制的阐明。目标1将 测试CB 1 R选择性偶联至特定Ga i/o蛋白以诱导神经元中ERK和FAK Tyr磷酸化的假设。目的2将检验CB 1 R介导的ERK和FAK Tyr 神经元细胞中的磷酸化涉及（1）特异性受体酪氨酸激酶（血管内皮生长因子受体、表皮生长因子受体）的反式激活，（2）MAPK磷酸酶的激活，和（3）神经祖细胞的神经元分化的诱导。为了完成本提案中概述的研究，将利用细胞培养的体外模型、特异性激酶和磷酸酶抑制剂、siRNA技术以及由LI-COR Biosciences开发的新型高通量In-Cell Western测定。此外，将使用已经用Ga i1 c351 g、Gai 2c 352 g、Gai 3c 351 g和Gao c351 g的质粒稳定转染的N18细胞，所述质粒赋予百日咳毒素对G1亚基的抗性。百日咳毒素将用于急性灭活Ga i/o亚型，这将允许CB 1 R与单个百日咳毒素抗性G1 c351/352 g i/o i/o亚型偶联，并且可以确定这种特异性Ga i/o蛋白在CB 1 R介导的ERK/FAK活化中所起的作用。从公共卫生的角度来看，内源性大麻素系统是治疗疼痛、成瘾和神经退行性疾病的药物治疗的靶点。蛋白激酶、蛋白磷酸酶和RTK反式激活在内源性大麻素介导的信号传导网络中发挥核心作用，这些信号传导网络调节细胞行为，并且也代表疾病中的潜在药物靶点。这些研究将有助于越来越多的证据表明，CB 1 R激动剂和拮抗剂在治疗慢性乙型肝炎中具有治疗益处。 药物滥用和神经变性疾病。 公共卫生相关性：内源性大麻素系统是开发治疗疼痛、成瘾和神经退行性疾病的药物的靶点。蛋白激酶、蛋白磷酸酶和GPCR介导的RTK反式激活在调节细胞行为的复杂信号网络中发挥核心作用，并代表疾病中的潜在药物靶标。从公共卫生的角度来看，这些研究将有助于越来越多的证据表明，CB 1受体激动剂和拮抗剂在调节涉及突触可塑性和神经元重塑的病理学，如药物滥用和神经退行性疾病的细胞过程中具有治疗益处。

DESCRIPTION (provided by applicant): This Kirschstein-NRSA Postdoctoral Fellowship (F32) will provide George Dalton with research training under the guidance of Dr. Allyn Howlett in the Department of Physiology and Pharmacology at Wake Forest University Health Sciences that will allow him to develop into a successful, independent scientist. The long- term goal of the proposed research in this application is to characterize the CB1receptor (CB 1R)-mediated signaling events that regulate the tyrosine (Tyr) phosphorylation of proteins in neurons. Dr. Dalton has established that cannabinoid agonists activate CB1Rs to induce the Tyr phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) in N18TG2 (N18) neuroblastoma cells. These findings form the basis for the investigation of two specific aims in this proposal that will involve the elucidation of the mechanisms by which CB1Rs regulate the Tyr phosphorylation of FAK and ERK in N18 cells. Aim 1 will test the hypothesis that CB1Rs couple selectively to specific Ga i/o proteins to induce ERK and FAK Tyr phosphorylation in neurons. Aim 2 will test the hypothesis that CB1R-mediated ERK and FAK Tyr phosphorylation in neuronal cells involves (1) the transactivation of specific receptor tyrosine kinases (vascular endothelial growth factor receptor, epidermal growth factor receptor), (2) the activation of MAPK phosphatases, and (3) induction of neuronal differentiation of neuroprogenitor cells. To accomplish the studies outlined in this proposal, in vitro models of cell culture, specific kinase and phosphatase inhibitors, siRNA technology, and a novel high throughput In-Cell Western assay developed by LI-COR Biosciences will be utilized. In addition, N18 cells will be used that have been stably transfected with plasmids for Ga i1c351g, Gai2c352g, Gai3c351g, and Gao c351g that confer pertussis toxin resistance to the G1 subunit. Pertussis toxin will be used to acutely inactivate Ga i/o subtypes which will allow CB1Rs to couple to a single pertussis toxin-resistant G1 c351/352g i/o i/o subtype and the role that this specific Ga i/o protein plays in CB1R-mediated ERK/FAK activation can be determined. From a public health perspective, the endocannabinoid system is a target for pharmacotherapy for the treatment of pain, addictions, and neurodegenerative disorders. Protein kinases, protein phosphatases, and RTK transactivation play a central role in the endocannabinoid-mediated signaling networks that regulate cellular behavior and also represent potential drug targets in disease. These studies will contribute to the growing body of evidence that CB1R agonists and antagonists have therapeutic benefits in the treatment of substance abuse and neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: The endocannabinoid system is a target for the development of drugs that treat pain, addictions, and neurodegenerative disorders. Protein kinases, protein phosphatases, and GPCR-mediated RTK transactivation play a central role in the complex signaling networks that regulate cellular behavior and represent potential drug targets in disease. From a public health perspective, these studies will contribute to the growing body of evidence that CB1 receptor agonists and antagonists have therapeutic benefits in modulating cellular processes that involve synaptic plasticity and neuronal remodeling in pathologies such as substance abuse and neurodegenerative diseases.