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

基本信息

批准号：
7803728
负责人：
ALLYN C HOWLETT
金额：
$ 18.43万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-10 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7803728
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

项目摘要

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）1型受体（CB1）介导δ-9-四氢大麻酚（大麻中的主要活性成分）的精神作用。 CB1 受体还介导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、疼痛感知、代谢稳态以及药物奖励和渴望方面发挥着重要作用。 CB1受体可以通过翻译后修饰和蛋白质-蛋白质相互作用进行调节，这可以改变这些受体的功能活性、细胞定位和表达水平。这些过程在限制 CB 激动剂的作用持续时间以及对重复施用 CB 激动剂产生耐受或依赖性方面发挥作用。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组成性（基础）活性。初步研究结果还表明，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 受体下调或脱敏，并且 CRIP1a 与 CB1 受体共定位，特别是在 CNS 谷氨酸能神经元中。提出以下具体目标来研究 CRIP1a 的功能：1) 开发新的细胞系和 siRNA 构建体作为工具，以确定 CRIP1a 对 CB1 受体急性和慢性激活的影响；2) 开发 CRIP1a 敲除小鼠系作为新的研究 CRIP1a 对体内生理功能、行为和 CB 药理学影响的工具。生化和细胞成像方法将用于确定细胞模型中 CRIP1a 共表达或 siRNA 介导的敲低对 CB1 受体介导的 G 蛋白关联（免疫共沉淀）和激活（GTP3S 结合）的影响，以及与调节蛋白2-抑制蛋白。然后将在这些细胞模型中检查 CRIP1a 对 CB1 受体脱敏、下调和内化的影响。将使用“flox”方法创建 CRIP1a 基因敲除小鼠品系。敲除小鼠将接受基本健康评估和体内表型分析，然后在体温过低、运动不足、僵直和抗伤害测试中确定敲除对 CB 激动剂药理效力的影响。然后将进行解剖学和生化研究，以确定 CRIP1a 敲除对 CB1 受体水平、G 蛋白激活和中枢神经系统细胞定位的影响。这些研究将提供有关 CRIP1a 在调节与动物功能反应相关的 CB1 受体介导的信号转导中的作用的有价值的数据。这项工作将为开发选择性调节 CB1 受体活性的药物提供新的靶标线索，以治疗药物成瘾和其他以内源性大麻素系统为关键调节成分的疾病。公共卫生相关性：CB1 大麻素受体介导大麻的许多作用，并与大脑中天然存在的大麻样物质相互作用。该系统对于食欲、疼痛感知、记忆、运动和协调的调节很重要，并且似乎在几种成瘾药物的奖励作用中发挥着作用。拟议的项目将研究一种新发现的蛋白质，称为 CRIP1a，它与 CB1 受体相互作用并似乎调节其功能。这些研究将使用转基因培养细胞系和 CRIP1a 基因已失活的小鼠来研究 CRIP1a 在 CB1 受体调节中的作用，以增加我们对大麻对大脑影响的了解，并可能为开发选择性调节 CB1 受体活性的药物。