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

描述(由申请人提供)：这项Kirschstein-NRSA博士后奖学金(F32)将为George Dalton提供在维克森林大学健康科学系生理学和药理学系Allyn Howlett博士的指导下的研究培训，使他能够发展成为一名成功的、独立的科学家。这项应用研究的长期目标是确定CB1受体(CB1R)介导的信号事件的特征，该信号事件调节神经元中蛋白质的酪氨酸(Tyr)磷酸化。道尔顿博士已经证实，大麻类激动剂可以激活CB1Rs，诱导N18TG2(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。这些发现为研究这一建议中的两个特定目标奠定了基础，这两个目标将涉及阐明CB1Rs调节N18细胞中FAK和ERK的Tyr磷酸化的机制。目的1验证CB1Rs选择性地与特定的Gai/o蛋白偶联以诱导神经元ERK和FAK Tyr磷酸化的假说。目的2验证CB1R介导的ERK和FAK Tyr在神经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，以及(3)诱导神经前体细胞的神经元分化。为了完成这项建议中概述的研究，将使用细胞培养的体外模型、特定的激酶和磷酸酶抑制剂、siRNA技术和Li-COR Biosciences开发的一种新的高通量In-cell Western分析。此外，N18细胞将被使用，该细胞已经稳定地转染了GA i1c351G、GaI 2c352G、GaI 3c351G和GaO C351G的质粒，这些载体赋予了对G1亚基的百日咳毒素抗性。百日咳毒素将被用来急性灭活GaI/o亚型，这将使CB1Rs与单个抗百日咳毒素的G1c351/352Gi/o亚型偶联，并确定这种特定的Gai/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调控细胞行为的信号网络中发挥着核心作用，也是治疗疾病的潜在药物靶点。这些研究将有助于越来越多的证据表明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。 公共卫生相关性：内源性大麻素系统是治疗疼痛、成瘾和神经退行性疾病药物开发的靶点。蛋白激酶、蛋白磷酸酶和GPCR介导的RTK反式激活在调控细胞行为的复杂信号网络中发挥着核心作用，并代表着疾病的潜在药物靶点。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，CB1受体激动剂和拮抗剂在调节细胞过程方面具有治疗益处，这些过程涉及物质滥用和神经退行性疾病等病理过程中的突触可塑性和神经元重塑。

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.