Mapping mu agonist-induced receptor-protein interactions for OPRM1 7TM variants

项目总结/摘要 吗啡和大多数临床使用的阿片类镇痛药，以及海洛因，主要通过μ阿片类药物发挥作用 受体。单拷贝μ阿片受体基因（OPRM 1）经历了广泛的替代前mRNA 剪接，产生一系列从啮齿动物到人类都保守的剪接变体。一种类型的 剪接变体是全长7-跨膜（TM）C-末端变体，其除了 在细胞内C-末端尾部的尖端的序列。越来越多的证据支持药理学 这些7TM C-末端变体的重要性。几种体外细胞模型证明了在以下方面的功能差异： μ激动剂诱导的G蛋白偶联、磷酸化、内化和内吞后分选，以及 区域和细胞特异性表达。更重要的是，在体内功能的几个C-末端变异体， 最近在具有两种近交系小鼠背景的C-末端截短小鼠模型中发现。尤其是外显子 C57BL/6J品系中7（E7）相关的C-末端截短降低了吗啡耐受和奖赏， 改变身体依赖性，而E4相关的C-末端截短加速吗啡 耐受和减少吗啡依赖而不影响吗啡奖赏。这些研究一起 强调了这些C-末端剪接变异体在介导多核苷酸的不同作用中的功能重要性。 阿片类药物，并提供了一个令人信服的理由，以进一步探讨C-末端7TM剪接的分子机制 μ阿片样物质作用的变体，如本申请中提出的。我们假设不同的C末端 Oprm1全长7TM变体的序列在确定受体与 一组独特的蛋白质，无论是在基础状态或响应于μ激动剂，导致其独特的信号转导 路径和功能。在这个应用中，我们建议使用新开发的邻近依赖性生物素 用与串联质量标签（TMT）偶联的工程化抗坏血酸过氧化物酶（APEX 2）鉴定 蛋白质组学的方法，映射这些瞬时或动态受体蛋白质相互作用下的基础状态， 在OPRM 1-KD Be（2）C细胞和原代纹状体中， 来自Oprm1敲除小鼠的神经元。我们将比较两种E7相关的C-末端7TM变体 mMOR-1O和mMOR-1C具有独特的体外和体内药理学特征，与E4相关 mMOR-1和截短形式mE1/2/3，其缺乏E3下游的额外C-末端序列。我们 将使用以下方法验证APEX2-TMT研究中选定候选药物的物理和/或功能相互作用： 许多方法，如NanoLuc二进制技术（NanoBit）和RNAi。拟议的研究 有望揭示mu激动剂诱导的受体-蛋白质相互作用，信号传导和功能的新见解， C-末端剪接变体，并提供适用于所有G-蛋白偶联受体的通用方法。 大约12%的非嗅觉GPCR具有可选的C末端剪接变体， 这一应用可能具有非常广泛的影响。

Project Summary/Abstract Morphine and most clinically used opioid analgesics, as well as heroin, act primarily through mu opioid receptors. The single-copy mu opioid receptor gene (OPRM1) undergoes extensive alternative pre-mRNA splicing, generating an array of splice variants that are conserved from rodents to humans. One type of the splice variants are full-length 7-transmembrane (TM) C-terminal variants that are identical except for the sequences at the tip of the intracellular C-terminal tail. Increasing evidence supports the pharmacological importance of these 7TM C-terminal variants. Several in vitro cell models demonstrate functional differences in mu agonist-induced G protein coupling, phosphorylation, internalization and post-endocytic sorting, as well as region- and cell-specific expression. More importantly, in vivo functions of several C-terminal variants were recently revealed in C-terminal truncation mouse models with two inbred mouse background. Particularly, exon 7 (E7)-associated C-terminal truncation in C57BL/6J strain diminished morphine tolerance and reward without altering physical dependence, whereas the E4-associated C-terminal truncation accelerated morphine tolerance and reduced morphine dependence without affecting morphine reward. Together, these studies underscore the functional importance of these C-terminal splice variants in mediating the diverse actions of mu opioids, and provide a compelling rationale to further explore molecular mechanisms of C-terminal 7TM splice variants in mu opioid actions, as proposed in this application. We hypothesize that different C-terminal sequences of the Oprm1 full-length 7TM variants are important in determining interaction of a receptor with a unique set of proteins either at basal states or in response to mu agonists, leading to their distinct signaling pathways and functions. In this application we propose using newly developed proximity-dependent biotin identification with an engineered ascorbate peroxidase (APEX2) coupled with tandem mass tag (TMT) proteomics approach, to map these transient or dynamic receptor-protein interactions under both basal state and activated conditions in response to different mu agonists in OPRM1-KD Be(2)C cells and primary striatal neurons derived from Oprm1 knockout mice. We will compare two E7-associated C-terminal 7TM variants mMOR-1O and mMOR-1C that have unique in vitro and in vivo pharmacological profiles with E4-associated mMOR-1, and a truncated version, mE1/2/3, that lacks additional C-terminal sequences downstream of E3. We will validate physical and/or functional interactions of selected candidates from APEX2-TMT study using a number of approaches, such as NanoLuc Binary Technology (NanoBit) and RNAi. The proposed studies promise to reveal new insights into mu agonist-induced receptor-protein interactions, signaling and function of the C-terminal splice variants, and to provide a general approach applicable to all G-protein coupled receptors. With approximately 12% of non-olfactory GPCRs having alternative C-terminal splice variants, the results from this application may have a very broad impact.