Identification and Characterization of mu Opioid Receptor Interacting Proteins

mu 阿片受体相互作用蛋白的鉴定和表征

• 批准号: 7595559
• 负责人: ROBERT LEVENSON
• 金额: $ 30.62万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7595559
• 关键词: Address; Adenylate Cyclase; Affect; Analgesics; Antibodies; Biochemical; Brain; Buprenorphine; Cell secretion; Cells; Complex; Coupling; Dependence; Disruption; Dominant-Negative Mutation; Drosophila genus; Drug abuse; Etiology; Etorphine; Future; G-Protein-Coupled Receptors; Generations; Goals; Heroin; Human; Instruction; Linkage Disequilibrium; Mammalian Cell; Maps; Mass Spectrum Analysis; Mediating; Morphine; Mus; Numbers; Opiate Addiction; Opiates; Opioid; Opioid Receptor; Orthologous Gene; Peptides; Pharmaceutical Preparations; Play; Property; Protein Binding Domain; Protein Kinase C; Proteins; Proteomics; Public Health; Receptor Cell; Receptor Mediated Signal Transduction; Receptor Signaling; Regulation; Rodent; Role; Series; Signal Transduction; Signal Transduction Pathway; Site; Small Interfering RNA; Synaptophysin; System; Techniques; Testing; Transgenic Mice; Ubiquitin; Validation; Withdrawal; Yeasts; cDNA Library; clinically relevant; cohort; desensitization; design; drug of abuse; filamin; human RIPK1 protein; inclusion criteria; insight; knock-down; mu opioid receptors; novel; novel therapeutics; protein function; protein protein interaction; receptor; receptor expression; research study; response; therapeutic target; trafficking; yeast two hybrid system

The mu-opioid receptor (MOR) mediates most of the actions of morphine and other clinically relevant analgesics as well as drugs of abuse such as heroin. The proteomic and functional studies proposed in this application are designed to elucidate the protein components of MOR signaling complexes. Identification of MOR interacting proteins will allow us to test our hypothesis that protein-protein interactions play an important role in regulating the MOR signal transduction pathway. In Aim I, we will identify and characterize MOR interacting proteins. A split-ubiquitin screen we have performed identified several novel MORIPs including GPR177, the mammalian ortholog of Drosophila Wntless. To identify additional MORIPs, each of the intracellular domains of the human MOR will be used as bait to separately screen a human brain cDNA library. We also propose to utilize highly selective anti-MOR antibodies combined with proteomics and mass spectrometry to identify a spectrum of MORIPs from immunoprecipitated mouse brain lysates. The major goal in Aim 2 will be to validate the MOR-protein interactions identified in Aim I. Cellular colocalization studies will confirm whether or not the MOR and candidate interactors are expressed within the same cells and intracellular compartments. Pull-down and coimmunoprecipitation will substantiate the MOR-protein interaction, while deletion mapping will permit identification of sites within the proteins that are necessary for the interactions to occur. Preliminary studies we have so far performed indicate that GPR177 meets all inclusion criteria and appears to represent a bona-fide MORIP. The goal in Aim 3 is to understand the functional significance of MOR/GPR177 interaction. We will examine the requirement for MOR/GPR177 interaction in MOR trafficking, desensitization, and signal transduction. We will also examine the role of the MOR/GPR177 interaction in regulating Wnt2 secretion from cells. To accomplish this goal, we will determine whether disrupting the MOR/GPR177 interaction, by expressing dominant negative forms of GPR177 or by knocking down GPR177 expression, affects the functional properties of the MOR. Identification of MOR interacting proteins will provide new insights into the etiology of drug abuse and dependence. RELEVANCE (See instructions): This project seeks to identify and evaluate the function of proteins that interact with and regulate the muopioid receptor (MOR), the cell associated receptor that mediates most of the analgesic actions of morphine as well as drugs of abuse. Understanding the function of GPR177, a novel interacting protein we identified, is likely to provide new insight into the etiology of drug abuse and dependence. MOR-interacting proteins may also represent novel therapeutic targets for the treatment of these important public health problems.

μ-阿片受体（莫尔）介导吗啡的大部分作用和其他临床相关作用。 止痛剂以及滥用药物如海洛因。本文提出的蛋白质组学和功能研究 本申请旨在阐明莫尔信号传导复合物的蛋白质组分。鉴定 莫尔相互作用蛋白质将使我们能够测试我们的假设，蛋白质-蛋白质相互作用发挥作用， 在调节莫尔信号转导通路中起重要作用。在目标I中，我们将确定和描述 莫尔相互作用蛋白。我们进行的分裂泛素筛选鉴定了几种新的MORIPs 包括GPR 177，果蝇Wntless的哺乳动物直系同源物。为了确定更多的MORIP， 人莫尔的胞内结构域将被用作诱饵以分别筛选人脑cDNA 图书馆我们还建议利用高选择性抗MOR抗体结合蛋白质组学和质谱技术， 通过质谱法鉴定来自免疫沉淀的小鼠脑裂解物的MORIP谱。主要 目标2的目标是验证目标I中确定的MOR-蛋白质相互作用。细胞共定位 研究将确认莫尔和候选相互作用物是否在相同的细胞内表达 和细胞内区室。下拉和免疫共沉淀将证实MOR蛋白 相互作用，而缺失作图将允许鉴定蛋白质内的位点，这些位点是相互作用所必需的。 互动发生。我们迄今为止进行的初步研究表明，GPR 177符合所有 入选标准，似乎代表了真正的MORIP。目标3的目标是了解 莫尔/GPR 177相互作用的功能意义。我们将研究莫尔/GPR 177的要求 莫尔转运、脱敏和信号传导中的相互作用。我们亦会研究 莫尔/GPR 177相互作用调节Wnt 2从细胞分泌。为了实现这一目标，我们将确定 无论是通过表达显性负性形式的GPR 177或通过表达显性负性形式的GPR 177来破坏莫尔/GPR 177相互作用， 敲低GPR 177表达，影响莫尔的功能特性。莫尔的识别 相互作用的蛋白质将为药物滥用和依赖的病因学提供新的见解。 相关性（参见说明）： 该项目旨在鉴定和评估与μ阿片样物质相互作用并调节μ阿片样物质的蛋白质的功能。 受体（莫尔），介导吗啡的大部分镇痛作用的细胞相关受体 以及滥用药物。了解GPR 177的功能，我们发现了一种新的相互作用蛋白， 可能为药物滥用和依赖的病因学提供新的见解。MOR相互作用蛋白 也可能是治疗这些重要公共卫生问题的新的治疗靶点。