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

描述（由申请人提供）：该Kirschstein-NRSA博士后奖学金（F32）将为George Dalton提供在Wake Forest大学健康科学系生理学和药理学博士Allyn Howlett指导下的研究培训，使他成为一名成功的独立科学家。本研究的长期目标是表征cb1受体（cb1r）介导的信号事件，这些信号事件调节神经元中酪氨酸（Tyr）蛋白的磷酸化。Dalton博士已经证实，大麻素激动剂激活CB1Rs，诱导N18TG2 （N18）神经母细胞瘤细胞中局灶黏附激酶（FAK）和细胞外信号调节激酶（ERK）的Tyr磷酸化。这些发现构成了本提案中两个特定目标的研究基础，这将涉及阐明CB1Rs调节N18细胞中FAK和ERK的Tyr磷酸化的机制。目的1将验证CB1Rs选择性地与特异性Ga i/o蛋白偶联以诱导神经元中ERK和FAK Tyr磷酸化的假设。Aim 2将验证cb1r介导的神经细胞ERK和FAK Tyr磷酸化的假设，包括(1)特定受体酪氨酸激酶（血管内皮生长因子受体、表皮生长因子受体）的反激活，(2)MAPK磷酸酶的激活，以及(3)神经祖细胞的神经元分化诱导。为了完成本提案中概述的研究，将利用细胞培养体外模型，特定激酶和磷酸酶抑制剂，siRNA技术和LI-COR生物科学公司开发的新型高通量细胞内Western测定。此外，N18细胞将被用于稳定转染了gaic1c351g、Gai2c352g、Gai3c351g和Gao c351g的质粒，这些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CB1Rs与单一百日咳毒素抗性G1 c351/352g i/o i/o亚型结合，并且可以确定这种特异性Ga i/o蛋白在cb1r介导的ERK/FAK激活中所起的作用。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目标。蛋白激酶、蛋白磷酸酶和RTK转激活在内源性大麻素介导的信号网络中发挥核心作用，这些信号网络调节细胞行为，也代表疾病中潜在的药物靶点。这些研究将有助于提供越来越多的证据，证明CB1R激动剂和拮抗剂在治疗药物滥用和神经退行性疾病方面具有治疗益处。

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.