CB1 Receptor Regulation by Cannabinoid Receptor Interacting Protein CRIP1a

大麻素受体相互作用蛋白 CRIP1a 对 CB1 受体的调节

• 批准号: 7667661
• 负责人: ALLYN C HOWLETT
• 金额: $ 23.57万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-10 至 2011-02-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7667661
• 关键词: 2-arachidonylglycerol; Acute; Agonist; Animals; Appetite Regulation; Arrestins; Attenuated; Behavior; Behavioral; Binding; Biochemical; Biological Models; Brain; C-terminal; CNR1 gene; Cannabinoids; Catalepsy; Cell Line; Cell model; Cells; Cerebellum; Chronic; Co-Immunoprecipitations; Data; Dependence; Development; Disease; Distal; Down-Regulation; Drug Addiction; Embryo; Endocannabinoids; Experimental Designs; Exposure to; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; Glutamates; Health; Homeostasis; Homer protein; Human; Immunoblotting; Immunohistochemistry; Investigation; Kidney; Knock-out; Knockout Mice; Ligands; Lipids; Marijuana; Mediating; Membrane Proteins; Memory; Metabolic; Metabotropic Glutamate Receptors; Microscopic; Modification; Motor Activity; Movement; Mus; Neuraxis; Neuroblastoma; Neuronal Plasticity; Neurons; Pain; Perception; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphorylation Site; Physiological; Physiology; Play; Process; Proteins; Receptor Activation; Receptor Mediated Signal Transduction; Receptor Signaling; Regulation; Research; Research Personnel; Research Project Grants; Rewards; Rodent Model; Role; Services; Short-Term Memory; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Specificity; System; Tail; Testing; Tetrahydrocannabinol; Tissues; Transgenic Mice; Translating; Vertebrates; Work; Yeasts; anandamide; arrestin 2; cannabinoid receptor; cellular imaging; craving; desensitization; drug development; drug reward; experience; genetic regulatory protein; high risk; human RIPK1 protein; in vivo; knock-down; natural hypothermia; neurotransmitter release; novel; overexpression; protein activation; protein protein interaction; public health relevance; receptor; receptor downregulation; receptor expression; receptor function; recombinase; response; small hairpin RNA; tool; trafficking; voltage; yeast two hybrid system

DESCRIPTION (provided by applicant): Cannabinoid (CB) type-1 receptors (CB1) in the central nervous system (CNS) mediate the psychoactive effects of delta-9-tetrahydrocannabinol, the major active constituent in marijuana. CB1 receptors also mediate many effects of the lipid-derived endogenous cannabinoids (endocannabinoids). This endocannabinoid system plays important roles in regulating motor activity and coordination, short-term memory, pain perception, metabolic homeostasis and drug reward and craving. CB1 receptors can be regulated by post-translational modification and protein-protein interactions, which can alter functional activity, cellular localization and expression levels of these receptors. These processes play a role in limiting the duration of action of CB agonists and in the development of tolerance or dependence upon repeated administration of CB agonists. The proposed project will investigate the function of a newly discovered CB receptor-interacting protein, CRIP1a, which binds to the distal C-terminus of CB1 receptors and attenuates constitutive (basal) activity of these receptors. Preliminary findings also suggest that CRIP1a can alter agonist-induced CB1 signaling in a ligand- and signaling pathway-dependent manner. Preliminary data indicate that CRIP1a can inhibit agonist- induced downregulation or desensitization of CB1 receptors, and that CRIP1a is co-localized with CB1 receptors, particularly in CNS glutamatergic neurons. The following specific aims are proposed to investigate the function of CRIP1a: 1) develop novel cell lines and siRNA constructs as tools to determine the effects of CRIP1a on acute and chronic activation of CB1 receptors and 2) develop a CRIP1a knockout mouse line as a novel tool to investigate effects of CRIP1a on physiological function, behavior and CB pharmacology in vivo. Biochemical and cell imaging approaches will be used to determine effects of co-expression or siRNA-mediated knockdown of CRIP1a in cell models on CB1 receptor-mediated G-protein association (co-immunoprecipitation) and activation (GTP3S binding), and interaction with the regulatory protein 2-arrestin. Effects of CRIP1a on CB1 receptor desensitization, downregulation and internalization will then be examined in these cell models. A CRIP1a gene knockout mouse line will be created using a "flox" approach. Knockout mice will be subjected to basic health assessment and in vivo phenotyping, followed by determination of effects of the knockout on the pharmacological potency of CB agonists in tests of hypothermia, hypolocomotion, catalepsy and antinociception. Anatomical and biochemical studies will then be conducted to determine effects of CRIP1a knockout on CB1 receptor levels, G-protein activation and cellular localization in the CNS. These studies will provide valuable data concerning the role of CRIP1a in the regulation of CB1 receptor-mediated signal transduction associated with functional responses in animals. This work will provide novel target leads for development of drugs that selectively regulate the activity of CB1 receptors for the treatment of drug addiction and other diseases in which the endocannabinoid system is a critical modulatory component. PUBLIC HEALTH RELEVANCE: CB1 cannabinoid receptors mediate many of the effects of marijuana and interact with naturally occurring marijuana-like substances in the brain. This system is important in the regulation of appetite, pain perception, memory, movement and coordination, and seems to play a role in the rewarding effects of several addictive drugs. The proposed project would study a newly discovered protein, called CRIP1a, which interacts with CB1 receptors and appears to modulate their function. These studies will investigate the role of CRIP1a in the regulation of CB1 receptors using genetically modified cultured cell lines and mice in which the CRIP1a gene has been inactivated, to increase our understanding of the effects of marijuana in the brain and perhaps provide a novel target for development of drugs that selectively regulate the activity of CB1 receptors.

描述（由申请人提供）：中枢神经系统（CNS）中的大麻素（CB）类型1受体（CB1）介导Delta-9-9-四氢大麻酚的精神活性，这是大麻中的主要活性成分。 CB1受体还介导了脂质衍生的内源性大麻素（内源性大麻素）的许多作用。这种内源性大麻素系统在调节运动活动和协调，短期记忆，疼痛感，代谢稳态以及药物奖励和渴望中起着重要作用。 CB1受体可以通过翻译后修饰和蛋白质 - 蛋白质相互作用来调节，这些相互作用可以改变功能活性，细胞定位和这些受体的表达水平。这些过程在限制CB激动剂的作用持续时间以及对CB激动剂反复给药的依赖性或依赖性的发展中发挥了作用。拟议的项目将研究新发现的CB受体相互作用蛋白CRIP1A的功能，该蛋白与CB1受体的远端C末端结合，并减弱这些受体的本构（基础）活性。初步发现还表明，CRIP1A可以以配体和信号通路依赖性方式改变激动剂诱导的CB1信号传导。初步数据表明，CRIP1A可以抑制激动剂诱导的CB1受体的下调或脱敏，并且CRIP1A与CB1受体共定位，尤其是在CNS谷氨酸能神经元中。提出了以下具体目的来研究CRIP1A的功能：1）开发新型的细胞系和siRNA构建体，作为确定CRIP1A对CB1受体急性和长期激活的影响的工具，以及2）开发CRIP1A敲除小鼠系列作为研究CRIP1A对生理功能和VIV中CB Prabricatic in Vivo的影响的新工具。生物化学和细胞成像方法将用于确定CB1受体介导的G蛋白缔合（共免疫沉淀）和激活（GTP3S结合）的共表达或siRNA介导的CRIP1A敲低CRIP1A的敲低以及与调节蛋白2- 2次寄生的调节蛋白的相互作用。在这些细胞模型中，将检查CRIP1A对CB1受体脱敏，下调和内在化的影响。将使用“ Flox”方法创建CRIP1A基因敲除鼠标线。敲除小鼠将接受基本的健康评估和体内表型，然后确定基因敲除对CB激动剂在低温，降低症，衰减和抗心敏度测试中的药理效力的影响。然后将进行解剖学和生化研究，以确定CRIP1A敲除对CNS中CB1受体水平，G蛋白激活和细胞定位的影响。这些研究将提供有关CRIP1A在调节CB1受体介导的与动物功能反应相关的CB1受体介导的信号转导的作用的有价值的数据。这项工作将为开发药物的开发提供新的靶标，这些药物有选择地调节CB1受体的活性来治疗药物成瘾和其他疾病，其中内源性大麻素系统是关键的调节成分。公共卫生相关性：CB1大麻素受体介导了大麻的许多作用，并与大脑中天然发生的大麻样物质相互作用。该系统在调节食欲，疼痛感，记忆，运动和协调方面很重要，并且似乎在几种成瘾性药物的奖励作用中起作用。拟议的项目将研究一种新发现的蛋白质，称为CRIP1A，该蛋白质与CB1受体相互作用并似乎在调节其功能。这些研究将研究CRIP1A在CB1受体调节CB1受体中的作用，其中CRIP1A基因已被灭活，以增强我们对大麻在大脑中的作用的理解，并可能为选择性地调节CB1受体活性的药物开发的新目标提供了新的靶场。