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.

MU-阿片受体（MOR）介导吗啡和其他临床相关的大多数作用 镇痛药以及海洛因等滥用药物。在此提出的蛋白质组学和功能研究 应用旨在阐明MOR信号复合物的蛋白质成分。识别 MOR相互作用的蛋白质将使我们能够测试蛋白质 - 蛋白质相互作用的假设 在调节MOR信号转导途径中的重要作用。在目标一世中，我们将确定和特征 MOR相互作用的蛋白质。我们进行的一个分裂泛素屏幕确定了几个新型摩擦 包括GPR177，果蝇的哺乳动物直系同源物。为了识别其他摩托车，每一个 人类MOR的细胞内结构域将用作诱饵，以分别筛选人脑cDNA 图书馆。我们还建议利用高度选择性的抗MOR抗体结合蛋白质组学和质量 光谱法鉴定了来自免疫沉淀的小鼠脑裂解物的MORIPS。专业 目标2的目标是验证AIM I中鉴定的Mor蛋白相互作用。细胞共定位 研究将确认MOR和候选者是否在同一细胞中表达 和细胞内室。下拉和共免疫沉淀将证实Mor-Protein 相互作用，而删除映射将允许识别蛋白质中所需的位点 发生的相互作用。到目前为止，我们进行的初步研究表明GPR177符合所有 纳入标准，似乎代表了善意的莫利普。目标3的目标是了解 MOR/GPR177相互作用的功能意义。我们将研究MOR/GPR177的要求 MOR运输，脱敏和信号转导的相互作用。我们还将研究 MOR/GPR177在调节细胞中Wnt2分泌的相互作用。为了实现这一目标，我们将确定 无论是通过表达GPR177的主要负面形式还是通过 击倒GPR177表达，会影响MOR的功能特性。 MOR的识别 相互作用的蛋白质将为药物滥用和依赖性的病因提供新的见解。 相关性（请参阅说明）： 该项目旨在识别和评估与植物相互作用并调节植物的蛋白质功能 受体（MOR），介导吗啡的大多数镇痛作用的细胞相关受体 以及滥用药物。了解GPR177的功能，我们确定的一种新型相互作用的蛋白质， 可能会提供有关药物滥用和依赖性病因的新见解。 mor互动蛋白 也可能代表用于治疗这些重要公共卫生问题的新型治疗靶标。