Department of Neurology and the Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York.
Department of Neurology and the Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York
Mol Pharmacol. 2020 Oct;98(4):518-527. doi: 10.1124/mol.120.119453. Epub 2020 Jul 28.
The -opioid receptor gene undergoes extensive alternative splicing to generate an array of splice variants. One group of splice variants excludes the first transmembrane (TM) domain and contains six TM domains. These 6TM variants are essential for the action of a novel class of analgesic drugs, including 3-iodobenzoyl-6-naltrexamide, which is potent against a spectrum of pain models without exhibiting the adverse side effects of traditional opiates. The 6TM variants are also involved in analgesic action through other drug classes, including -opioid and opioids and -adrenergic drugs. Of the five 6TM variants in mouse, mouse -opioid receptor (mMOR)-1G is abundant and conserved from rodent to human. In the present study, we demonstrate a new function of mMOR-1G in enhancing expression of the full-length 7TM -opioid receptor, mMOR-1. When coexpressed with mMOR-1 in a Tet-Off inducible CHO cell line, mMOR-1G has no effect on mMOR-1 mRNA expression but greatly increases mMOR-1 protein expression in a dose-dependent manner determined by opioid receptor binding and [S] guanosine 5'-3--(thio)triphosphate binding. Subcellular fractionation analysis using OptiPrep density gradient centrifugation shows an increase of functional mMOR-1 receptor in plasma membrane-enriched fractions. Using a coimmunoprecipitation approach, we further demonstrate that mMOR-1G physically associates with mMOR-1 starting at the endoplasmic reticulum, suggesting a chaperone-like function. These data provide a molecular mechanism for how mMOR-1G regulates expression and function of the full-length 7TM -opioid receptor. SIGNIFICANCE STATEMENT: The current study establishes a novel function of mouse -opioid receptor (mMOR)-1G, a truncated splice variant with six transmembrane (TM) domains of the mouse -opioid receptor gene, in enhancing expression of the full-length 7TM mMOR-1 through a chaperone-like function.
-阿片受体基因经历广泛的选择性剪接，产生一系列剪接变体。一组剪接变体排除了第一个跨膜（TM）结构域，并包含六个 TM 结构域。这些 6TM 变体是一类新型阿片类镇痛药发挥作用的必要条件，包括 3-碘苯甲酰基-6-纳曲胺，它对一系列疼痛模型有效，而没有表现出传统阿片类药物的不良反应。这些 6TM 变体也通过其他药物类别参与镇痛作用，包括 -阿片受体和阿片类药物和 -肾上腺素能药物。在小鼠的五种 6TM 变体中，小鼠 -阿片受体（mMOR）-1G 丰富且从啮齿动物到人保持保守。在本研究中，我们证明了 mMOR-1G 在增强全长 7TM -阿片受体，mMOR-1 的表达方面的新功能。当在 Tet-Off 可诱导的 CHO 细胞系中与 mMOR-1 共表达时，mMOR-1G 对 mMOR-1 mRNA 表达没有影响，但以剂量依赖的方式大大增加 mMOR-1 蛋白表达，这是通过阿片受体结合和 [S]鸟苷 5'-3--（硫）三磷酸结合来确定的。使用 OptiPrep 密度梯度离心的亚细胞部分分析显示，在富含质膜的级分中增加了功能性 mMOR-1 受体。通过共免疫沉淀方法，我们进一步证明 mMOR-1G 从内质网开始与 mMOR-1 物理结合，提示其具有伴侣样功能。这些数据为 mMOR-1G 如何调节全长 7TM -阿片受体的表达和功能提供了分子机制。意义声明：本研究确立了小鼠 -阿片受体（mMOR）-1G 的一个新功能，即小鼠 -阿片受体基因的一个截断的 6TM 变体，通过伴侣样功能增强全长 7TM mMOR-1 的表达。
