Identification and Characterization of mu Opioid Receptor Interacting Proteins

MU-阿片受体(MOR)介导了吗啡和其他临床相关物质的大部分作用 止痛药以及海洛因等滥用药物。本文提出的蛋白质组学和功能研究 应用的目的是阐明MOR信号复合体的蛋白质成分。身份识别 MOR相互作用的蛋白质将使我们能够检验我们的假设，即蛋白质-蛋白质相互作用在 在调节MOR信号转导通路中发挥重要作用。在目标一中，我们将确定和描述 MOR相互作用蛋白。我们已经进行的分裂泛素屏幕识别了几个新的MORIP 包括GPR177，果蝇Wnless的哺乳动物直系同源基因。要确定其他MORIP，每个 人MOR的胞内结构域将作为诱饵单独筛选人脑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 从免疫沉淀小鼠脑裂解物中鉴定MORIPs的光谱。少校 目标2的目标将是验证在目标1中确定的MOR-蛋白质相互作用。 研究将证实MOR和候选相互作用因子是否在同一细胞内表达 和细胞内的隔间。下拉和免疫共沉淀将证实MOR-蛋白 相互作用，而缺失作图将允许识别蛋白质内的必要位点 将要发生的相互作用。我们到目前为止进行的初步研究表明，GPR177符合所有 纳入标准，似乎代表了真正的MORIP。目标3的目标是理解 MOR/GPR177相互作用的功能意义。我们将研究MOR/GPR177的要求 吗啡转运、脱敏和信号转导中的相互作用。我们还将研究 MOR/GPR177相互作用调节细胞WNT2的分泌为了实现这一目标，我们将确定 无论是通过表达GPR177的显性否定形式还是通过表达MOR/GPR177相互作用 下调GPR177的表达，影响MOR的功能性质。更多信息的识别 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(请参阅说明)： 该项目致力于识别和评估与类穆阿片相互作用并调节其功能的蛋白质。 受体(MOR)，介导吗啡大部分镇痛作用的细胞相关受体 以及滥用药物。了解我们发现的一种新的相互作用蛋白GPR177的功能， 很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目标。

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.