Purification and Functional Reconstitution of Monomeric μ-Opioid Receptors ALLOSTERIC MODULATION OF AGONIST BINDING BY Gi2
Purification and Functional Reconstitution of Monomeric μ-Opioid Receptors ALLOSTERIC MODULATION OF AGONIST BINDING BY Gi2
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DOI：
10.1074/jbc.m109.026922
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发表时间：
2009-09-25
影响因子：
4.8
通讯作者：
Sunahara, Roger K.
中科院分区：
文献类型：
--
作者：
Kuszak, Adam J.;Pitchiaya, Sethuramasundaram;Sunahara, Roger K.
尽管对 mu-阿片受体 (MOR) 进行了广泛的表征，但分离受体的生化特性仍不清楚。根据最近的报道，我们提出 MOR 的单体形式可以激活 G 蛋白并受到变构调节。构建了与黄色荧光蛋白（YMOR）融合的μ阿片受体并在昆虫细胞中表达。 YMOR 以高亲和力结合配体，显示激动剂刺激的 [S-35]鸟苷 5'-（γ-硫代）三磷酸与 G α(i) 结合，并受到昆虫细胞膜中偶联的 G(i) 蛋白异三聚体的变构调节，以及以高密度脂蛋白颗粒形式重构为磷脂双层的纯化蛋白。荧光标记受体的单粒子成像表明重建的 YMOR 是单体的。此外，Cy3 标记的激动剂 [Lys(7)、Cys(8)]dermorphin 的单分子成像阐明了一种研究 G 蛋白偶联受体-配体结合的新方法，并表明每个单体 YMOR 结合一个激动剂分子。这些数据共同支持这样的观点，即激动剂和拮抗剂结合不需要μ阿片受体的寡聚化，并且单体受体是G蛋白激活和G蛋白对激动剂结合的强变构调节的最小功能单位。
Despite extensive characterization of the mu-opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A mu-opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [S-35]guanosine 5'-(gamma-thio)triphosphate binding to G alpha(i), and is allosterically regulated by coupled G(i) protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys(7), Cys(8)]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the mu-opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.