CB1 Receptor Regulation by Cannabinoid Receptor Interacting Protein CRIP1a

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)介导了大麻中主要活性成分三角洲-9-四氢大麻酚的精神作用。CB1受体还介导脂源性内源性大麻素(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、短时记忆、痛觉、代谢动态平衡以及药物奖赏和渴求等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-蛋白质相互作用来调节，从而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激动剂的作用时间和形成对CB激动剂反复给药的耐受性或依赖性方面发挥了作用。该项目将研究一种新发现的CB受体相互作用蛋白CRIP1a的功能，它与CB1受体的远端C末端结合，并减弱这些受体的结构性(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的方式改变激动剂诱导的CB1信号。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，尤其是在中枢谷氨酸能神经元。为了研究CRIP1a的功能，我们提出了以下具体目标：1)建立新的细胞系和siRNA结构，作为研究CRIP1a对CB1受体急性和慢性激活的影响的工具；2)建立CRIP1a基因敲除小鼠系，作为研究CRIP1a对体内生理功能、行为和CB药理学影响的新工具。生化和细胞成像方法将被用来确定在细胞模型中共表达或siRNA介导的CRIP1a对CB1受体介导的G蛋白结合(共免疫沉淀)和激活(GTP3S结合)以及与调节蛋白2-arrestin相互作用的影响。然后将在这些细胞模型中检测CRIP1a对CB1受体脱敏、下调和内化的影响。一个CRIP1a基因敲除小鼠品系将使用“FLOX”方法创建。基因敲除小鼠将接受基本的健康评估和体内表型鉴定，然后在低温、低血压、嗜睡和抗伤害作用测试中确定基因敲除对CB激动剂药理效力的影响。然后将进行解剖和生化研究，以确定CRIP1a基因敲除对CB1受体水平、G蛋白激活和中枢神经系统细胞定位的影响。这些研究将为CRIP1a在调节CB1受体介导的与动物功能反应相关的信号转导中的作用提供有价值的数据。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向，用于治疗药物成瘾和其他疾病，在这些疾病中，内源性大麻素系统是关键的调节成分。与公共卫生相关：CB1大麻素受体介导大麻的许多作用，并与大脑中自然产生的大麻样物质相互作用。这个系统在调节食欲、痛觉、记忆、运动和协调方面很重要，似乎在几种成瘾药物的奖赏效应中发挥了作用。这个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，似乎调节它们的功能。这些研究将利用转基因培养细胞系和CRIP1a基因失活的小鼠来研究CRIP1a在CB1受体调节中的作用，以增加我们对大麻在大脑中的影响的了解，并可能为选择性调节CB1受体活性的药物的开发提供一个新的靶点。

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.